Modulation of PPAR binding protein expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of PPAR binding protein. The compositions comprise oligonucleotides, targeted to nucleic acid encoding PPAR binding protein. Methods of using these compounds for modulation of PPAR binding protein expression and for diagnosis and treatment of disease associated with expression of PPAR binding protein are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods for modulating the expression of PPAR binding protein. In particular, this invention relates to compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding PPAR binding protein. Such compounds are shown herein to modulate the expression of PPAR binding protein.

BACKGROUND OF THE INVENTION

[0002] Steroid, thyroid and retinoid hormones produce a diverse array of physiologic effects through the regulation of gene expression. Upon entering the cell, these hormones bind to a unique group of intracellular nuclear receptors which have been characterized as ligand-dependent transcription factors. This complex then moves into the nucleus where the receptor and its cognate ligand interact with the transcription preinitiation complex affecting its stability and ultimately, the rate of transcription of the target genes. Members of the nuclear receptor family share several structural features including a central, highly conserved DNA-binding domain which targets the receptor to specific DNA sequences known as hormone response elements (Kliewer et al., Science, 1999, 284, 757-760).

[0003] The thyroid hormone receptors (TRs) are hormone-dependent transcription factors that regulate expression of a variety of specific target genes. They must specifically interact with a number of proteins as they progress from their initial translation and nuclear translocation to heterodimerization with retinoid X receptors (RXRs), functional interactions with other transcription factors and the basic transcriptional apparatus, and eventually, degradation.

[0004] Human PPAR binding protein (peroxisome proliferator activated receptor binding protein, also known as PBP, CRSP1, CRSP200, DRIP230, DRIP205, TRAP220, PRIP and thyroid hormone receptor interactor 2) was identified by Drane et al. in 1997. It was demonstrated that the gene is a regulator of p53, a well known transcription repressor implicated in many cancers (Drane et al., Oncogene, 1997, 15, 3013-3024). The gene is expressed as an 8.5 kb transcript in many tissues with highest levels observed in heart (Drane et al., Oncogene, 1997, 15, 3013-3024) and has been localized to chromosome 17q12-q21.1 (Frade et al., Cancer Res., 2000, 60, 6585-6589). Subsequent investigations indicated that PPAR binding protein downregulates p53-dependent apoptosis (Frade et al., Oncogene, 2002, 21, 861-866).

[0005] Isolated nucleic acid sequences encoding PPAR binding protein are disclosed and claimed in U.S. Pat. No. 6,248,520 and corresponding PCT publication WO 00/01820 (Roeder et al., 2000; Roeder et al., 2001).

[0006] Zhu et al. identified mouse PPAR binding protein and its interaction with peroxisome proliferator-activated receptor-gamma (PPARG), a multifunctional transcription factor which plays a central role in the control of insulin sensitivity via the regulation of adipocyte gene expression and differentiation (Zhu et al., Proc. Natl. Acad. Sci. U.S. A., 1999, 96, 10848-10853).

[0007] Ablation of the murine PPAR binding protein gene causes death in the early gestational stage with heart failure, impaired neuronal development and placental vasculature and extensive apoptosis (Ito et al., Mol. Cell, 2000, 5, 683-693; Zhu et al., J. Biol. Chem., 2000, 275, 14779-14782).

[0008] Kang et al. have identified interactions of PPAR binding protein with estrogen receptors alpha and beta which directly enhance estrogen receptor function in vitro (Kang Yun et al., Proc. Natl. Acad. Sci. U.S. A., 2002, 99, 26422647).

[0009] Disclosed and claimed in PCT publication WO 99/31231 is a pharmaceutical composition comprising an antisense sequence capable of specifically hybridizing with a nucleic acid sequence of PPAR binding protein, in association with a pharmaceutically acceptable carrier (Frade, 1999).

[0010] PPAR binding protein may prove to be a potentially useful strategy for therapeutic intervention in metabolic disorders such as obesity and diabetes and in hyperproliferative disorders such as cancers which arise from aberrant apoptosis.

[0011] Currently, there are no known therapeutic agents that effectively inhibit the synthesis of PPAR binding protein. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting PPAR binding protein function.

[0012] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of PPAR binding protein expression.

[0013] The present invention provides compositions and methods for modulating PPAR binding protein expression.

SUMMARY OF THE INVENTION

[0014] The present invention is directed to compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding PPAR binding protein, and which modulate the expression of PPAR binding protein. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of PPAR binding protein and methods of modulating the expression of PPAR binding protein in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention. Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of PPAR binding protein are also set forth herein. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0015] A. Overview of the Invention

[0016] The present invention employs compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding PPAR binding protein. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding PPAR binding protein. As used herein, the terms “target nucleic acid” and “nucleic acid molecule encoding PPAR binding protein” have been used for convenience to encompass DNA encoding PPAR binding protein, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of this invention with its target nucleic acid is generally referred to as “antisense”. Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as “antisense inhibition.” Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.

[0017] The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of PPAR binding protein. In the context of the present invention, “modulation” and “modulation of expression” mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.

[0018] In the context of this invention, “hybridization” means the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

[0019] An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

[0020] In the present invention the phrase “stringent hybridization conditions” or “stringent conditions” refers to Conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.

[0021] “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.

[0022] It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the antisense compounds of the present invention comprise at least 70% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise 90% sequence complementarity and even more preferably comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0023] B. Compounds of the Invention

[0024] According to the present invention, compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid. One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.

[0025] While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing.

[0026] The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from work in the nematode, Caenorhabditis elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown that the primary interference effects of dsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs which are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697).

[0027] In the context of this invention, the term “oligomeric compound” refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.

[0028] While oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.

[0029] The compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

[0030] In one preferred embodiment, the compounds of the invention are 12 to 50 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases in length.

[0031] In another preferred embodiment, the compounds of the invention are 15 to 30 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

[0032] Particularly preferred compounds are oligonucleotides from about 12 to about 50 nucleobases, even more preferably those comprising from about 15 to about 30 nucleobases.

[0033] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.

[0034] Exemplary preferred antisense compounds include oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.

[0035] C. Targets of the Invention

[0036] “Targeting” an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target nucleic acid encodes PPAR binding protein.

[0037] The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result within the context of the present invention, the term “region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid. “Sites,” as used in the present invention, are defined as positions within a target nucleic acid.

[0038] Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding PPAR binding protein, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

[0039] The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 31) from a translation termination codon. Consequently, the “start codon region” (or “translation initiation codon region”) and the “stop codon region” (or “translation termination codon region”) are all regions which may be targeted effectively with the antisense compounds of the present invention.

[0040] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a preferred region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

[0041] Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA (or corresponding nucleotides on the gene). The 5′ cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also preferred to target the 5′ cap region.

[0042] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.

[0043] It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence.

[0044] Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants”. Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0045] It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also preferred target nucleic acids.

[0046] The locations on the target nucleic acid to which the preferred antisense compounds hybridize are hereinbelow referred to as “preferred target segments.” As used herein the term “preferred target segment” is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.

[0047] While the specific sequences of certain preferred target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target segments may be identified by one having ordinary skill.

[0048] Target segments 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.

[0049] Target segments can include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.

[0050] Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0051] D. Screening and Target Validation

[0052] In a further embodiment, the “preferred target segments” identified herein may be employed in a screen for additional compounds that modulate the expression of PPAR binding protein. “Modulators” are those compounds that decrease or increase the expression of a nucleic acid molecule encoding PPAR binding protein and which comprise at least an 8-nucleobase portion which is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding PPAR binding protein with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding PPAR binding protein. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding PPAR binding protein, the modulator may then be employed in further investigative studies of the function of PPAR binding protein, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.

[0053] The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.

[0054] Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processsing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., Science, 2002, 295, 694-697).

[0055] The compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between PPAR binding protein and a disease state, phenotype, or condition. These methods include detecting or modulating PPAR binding protein comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of PPAR binding protein and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.

[0056] E. Kits, Research Reagents, Diagnostics, and Therapeutics

[0057] The compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.

[0058] For use in kits and diagnostics, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0059] As one nonlimiting example, expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0060] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0061] The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding PPAR binding protein. For example, oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective PPAR binding protein inhibitors will also be effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding PPAR binding protein and in the amplification of said nucleic acid molecules for detection or for use in further studies of PPAR binding protein. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding PPAR binding protein can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of PPAR binding protein in a sample may also be prepared.

[0062] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.

[0063] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of PPAR binding protein is treated by administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of a PPAR binding protein inhibitor. The PPAR binding protein inhibitors of the present invention effectively inhibit the activity of the PPAR binding protein protein or inhibit the expression of the PPAR binding protein protein. In one embodiment, the activity or expression of PPAR binding protein in an animal is inhibited by about 10%. Preferably, the activity or expression of PPAR binding protein in an animal is inhibited by about 30%. More preferably, the activity or expression of PPAR binding protein in an animal is inhibited by 50% or more.

[0064] For example, the reduction of the expression of PPAR binding protein may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding PPAR binding protein protein and/or the PPAR binding protein protein itself.

[0065] The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.

[0066] F. Modifications

[0067] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0068] Modified Internucleoside Linkages (Backbones)

[0069] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0070] Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 51-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0071] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0072] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0073] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0074] Modified Sugar and Internucleoside Linkages-Mimetics

[0075] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e. the backbone), of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate target nucleic acid. One such compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0076] Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0077] Modified Sugars

[0078] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0079] Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0080] A further preferred modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is preferably a methylene (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0081] Natural and Modified Nucleobases

[0082] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0083] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0084] Conjugates

[0085] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of which are incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0086] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0087] Chimeric Compounds

[0088] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.

[0089] The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNAseL which cleaves both cellular and viral RNA. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0090] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0091] G. Formulations

[0092] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0093] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0094] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0095] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0096] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[0097] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0098] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0099] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.

[0100] Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0101] Formulations of the present invention include liposomal formulations. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[0102] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0103] The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0104] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0105] One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0106] Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).

[0107] For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Topical formulations are described in detail in United States patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.

[0108] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.

[0109] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0110] Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0111] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0112] H. Dosing

[0113] The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0114] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1

[0115] Synthesis of Nucleoside Phosphoramidites The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine, 2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modified amidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate, 5′-O-DMT-21-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) nucleoside amidites, 2′-(Dimethylaminooxyethoxy) nucleoside amidites, 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine, 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine, 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine, 2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′ [(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′ dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites, 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine, 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine and 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2

[0116] Oligonucleotide and Oligonucleoside Synthesis

[0117] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0118] Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

[0119] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0120] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0121] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

[0122] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0123] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0124] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0125] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0126] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

[0127] Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0128] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0129] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 3

[0130] RNA Synthesis

[0131] In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5′-hydroxyl in combination with an acid-labile orthoester protecting group on the 2′-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2′ hydroxyl.

[0132] Following this procedure for the sequential protection of the 5′-hydroxyl in combination with protection of the 2′-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.

[0133] RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3′- to 5′-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5′-end of the first nucleoside. The support is washed and any unreacted 5′-hydroxyl groups are capped with acetic anhydride to yield 5′-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5′-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.

[0134] Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2′-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.

[0135] The 2′-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.

[0136] Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand., 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).

[0137] RNA antisense compounds (RNA oligonucleotides) of the present invention can be synthesized by the methods herein or purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized, complementary RNA antisense compounds can then be annealed by methods known in the art to form double stranded (duplexed) antisense compounds. For example, duplexes can be formed by combining 30 μl of each of the complementary strands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and 15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisense compounds can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid.

Example 4

[0138] Synthesis of Chimeric Oligonucleotides

[0139] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[0140] [2′-O-Me]-[2′-deoxy]—[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides

[0141] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 31 wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[0142] [2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0143] [2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

[0144] [2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxy Phosphorothioate]-[2′-O-(2-Methoxyethyl)Phosphodiester] Chimeric Oligonucleotides

[0145] [2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxy phosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0146] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5

[0147] Design and Screening of Duplexed Antisense Compounds Targeting PPAR Binding Protein

[0148] In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target PPAR binding protein. The nucleobase sequence of the antisense strand of the duplex comprises at least a portion of an oligonucleotide in Table 1. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini.

[0149] For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG and having a two-nucleobase overhang of deoxythymidine (dT) would have the following structure:   cgagaggcggacgggaccgTT Antisense Strand   ||||||||||||||||||| TTgctctccgcctgccctggc Complement

[0150] RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 μM. Once diluted, 30 uL of each strand is combined with 15 uL of a 5× solution of annealing buffer. The final concentration of said buffer is 100 mm potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL. This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 uM. This solution can be stored frozen (−20° C.) and freeze-thawed up to 5 times.

[0151] Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate PPAR binding protein expression.

[0152] When cells reached 80% confluency, they are treated with duplexed antisense compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at a final concentration of 200 nM. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by RT-PCR.

Example 6

[0153] Oligonucleotide Isolation

[0154] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32+/−48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171 Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

[0155] Oligonucleotide Synthesis—96 Well Plate Format

[0156] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0157] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

[0158] Oligonucleotide Analysis—96-Well Plate Format

[0159] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

[0160] Cell Culture and Oligonucleotide Treatment

[0161] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.

[0162] T-24 Cells:

[0163] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0164] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0165] A549 Cells:

[0166] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

[0167] NHDF Cells:

[0168] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

[0169] HEK Cells:

[0170] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0171] Treatment With Antisense Compounds:

[0172] When cells reached 65-75% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. Cells are treated and data are obtained in triplicate. After 4-7 hours of treatment at 37° C., the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0173] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10

[0174] Analysis of Oligonucleotide Inhibition of PPAR Binding Protein Expression

[0175] Antisense modulation of PPAR binding protein expression can be assayed in a variety of ways known in the art. For example, PPAR binding protein mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0176] Protein levels of PPAR binding protein can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to PPAR binding protein can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

Example 11

[0177] Design of Phenotypic Assays and In Vivo Studies for the Use of PPAR Binding Protein Inhibitors

[0178] Phenotypic Assays

[0179] Once PPAR binding protein inhibitors have been identified by the methods disclosed herein, the compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition. Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of PPAR binding protein in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).

[0180] In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with PPAR binding protein inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.

[0181] Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.

[0182] Analysis of the geneotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the PPAR binding protein inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.

[0183] In Vivo Studies

[0184] The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.

[0185] The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study. To account for the psychological effects of receiving treatments, volunteers are randomly given placebo or PPAR binding protein inhibitor. Furthermore, to prevent the doctors from being biased in treatments, they are not informed as to whether the medication they are administering is a PPAR binding protein inhibitor or a placebo. Using this randomization approach, each volunteer has the same chance of being given either the new treatment or the placebo.

[0186] Volunteers receive either the PPAR binding protein inhibitor or placebo for eight week period with biological parameters associated with the indicated disease state or condition being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements include the levels of nucleic acid molecules encoding PPAR binding protein or PPAR binding protein protein levels in body fluids, tissues or organs compared to pretreatment levels. Other measurements include, but are not limited to, indices of the disease state or condition being treated, body weight, blood pressure, serum titers of pharmacologic indicators of disease or toxicity as well as ADME (absorption, distribution, metabolism and excretion) measurements.

[0187] Information recorded for each patient includes age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for the indicated disease or condition.

[0188] Volunteers taking part in this study are healthy adults (age 18 to 65 years) and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and PPAR binding protein inhibitor treatment. In general, the volunteers treated with placebo have little or no response to treatment, whereas the volunteers treated with the PPAR binding protein inhibitor show positive trends in their disease state or condition index at the conclusion of the study.

Example 12

[0189] RNA Isolation

[0190] Poly(A)+ mRNA Isolation

[0191] Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0192] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

[0193] Total RNA Isolation

[0194] Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 μL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0195] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13

[0196] Real-Time Quantitative PCR Analysis of PPAR Binding Protein mRNA Levels

[0197] Quantitation of PPAR binding protein mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0198] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0199] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0200] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0201] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

[0202] Probes and primers to human PPAR binding protein were designed to hybridize to a human PPAR binding protein sequence, using published sequence information (a genomic sequence of human PPAR binding protein represented by the complement of residues 10000-58000 of GenBank accession number AC009283.2, incorporated herein as SEQ ID NO: 4). For human PPAR binding protein the PCR primers were: forward primer: AAAGCAGTAAATCAGAAGGTTCATCA (SEQ ID NO: 5) reverse primer: CCAGACTGGGATGAATTTTTGG (SEQ ID NO: 6) and the PCR probe was: FAM-TTCCAAGTTAAGTAGCAGTATGTATTCTAGC-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO:8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14

[0203] Northern Blot Analysis of PPAR Binding Protein mRNA Levels

[0204] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (StraLagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0205] To detect human PPAR binding protein, a human PPAR binding protein specific probe was prepared by PCR using the forward primer AAAGCAGTAAATCAGAAGGTTCATCA (SEQ ID NO: 5) and the reverse primer CCAGACTGGGATGAATTTTTGG (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0206] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

[0207] Antisense Inhibition of Human PPAR Binding Protein Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0208] In accordance with the present invention, a series of antisense compounds were designed to target different regions of the human PPAR binding protein RNA, using published sequences (a genomic sequence of human PPAR binding protein represented by the complement of residues 10000-58000 of GenBank accession number AC009283.2, incorporated herein as SEQ ID NO: 4 and GenBank accession number NM_(—)004774.1, incorporated herein as SEQ ID NO: 11). The compounds are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the compound binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human PPAR binding protein mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments in which A549 cells were treated with the oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”. TABLE 1 Inhibition of human PPAR binding protein mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET CONTROL SEQ ID TARGET SEQ ID SEQ ID ISIS # REGION NO SITE SEQUENCE % INHIB NO NO 241128 5′UTR 4 666 ctagatccgccacaaaagga 54 13 1 241129 Start 4 3878 gagagaactcatcttactca 19 14 1 Codon 241130 Coding 4 3904 ttaaattttgcatggagccg 47 15 1 241131 Coding 4 3946 tgacgcacaagcttaatggt 66 16 1 241132 Coding 4 8178 tccaaacagctgaccaaatg 73 17 1 241133 Coding 4 8183 atgtctccaaacagctgacc 60 18 1 241134 Coding 11 386 gttactttgagagccttctg 45 19 1 241135 Coding 11 391 aagatgttactttgagagcc 27 20 1 241136 Coding 4 11104 aaacgatcagtcattgctgg 35 21 1 241137 Coding 11 447 agagcccagtccattctgtc 57 22 1 241138 Coding 4 11265 tcagtgccactggcactgag 37 23 1 241139 Coding 4 11286 atatctgacgtgatgtaaca 50 24 1 241140 Coding 4 11291 agaacatatctgacgtgatg 46 25 1 241141 Coding 4 11296 cacatagaacatatctgacg 75 26 1 241142 Coding 4 11301 acttccacatagaacatatc 0 27 1 241143 Coding 4 11306 actgcacttccacatagaac 43 28 1 241144 Coding 4 11331 tcaoaaagctgtcctgcagg 47 29 1 241145 Coding 4 11355 tccccatggtgagccacttt 51 30 1 241146 Coding 4 11360 gattctccccatggtgagcc 51 31 1 241147 Coding 4 11365 cacaggattctccccatggt 63 32 1 241148 Coding 11 575 cagctcacaggattctcccc 73 33 1 241149 Coding 4 20867 gaagaatcttatccaaggga 0 34 1 241150 Coding 4 27330 gttccttcaattgtcactga 65 35 1 241151 Coding 11 1157 aatggaattcctgtgcagtt 23 36 1 241152 Coding 4 32297 tagttcttttgacacagctt 49 37 1 241153 Coding 4 41414 caccatgtcttcaactgtct 75 38 1 241154 Coding 4 41558 atctttgatgctcatgctca 66 39 1 241155 Coding 4 41622 tggtaagaattgggttctga 51 40 1 241156 Coding 4 41645 gttccctgtgatttgcaaca 54 41 1 241157 Coding 4 41650 cccccgttccctgtgatttg 73 42 1 241158 Coding 4 41666 cgagccaatggtagaccccc 85 43 1 241159 Coding 4 41671 ggactcgagccaatggtaga 46 44 1 241160 Coding 4 41676 gggtcggactcgagccaatg 40 45 1 241161 Coding 4 41709 tcgaagagacaggtggcggc 38 46 1 241162 Coding 4 41714 ggccatcgaagagacaggtg 37 47 1 241163 Coding 4 41719 ttgccggccatcgaagagac 19 48 1 241164 Coding 4 41724 tggtgttgccggccatcgaa 56 49 1 241165 Coding 4 41729 gttcttggtgttgccggcca 56 50 1 241166 Coding 4 41737 atcgggtggttcttggtgtt 49 51 1 241167 Coding 4 41742 tgagcatcgggtggttcttg 0 52 1 241168 Coding 4 41747 gttcatgagcatcgggtggt 66 53 1 241169 Coding 4 41752 agaaggttcatgagcatcgg 59 54 1 241170 Coding 4 41791 ccataaagggttgagaaatc 13 55 1 241171 Coding 4 41796 tgcttccataaagggttgag 63 56 1 241172 Coding 4 41802 aagggctgcttccataaagg 51 57 1 241173 Coding 4 41807 ttctaaagggctgcttccat 63 58 1 241174 Coding 4 41812 tgcctttctaaagggctgct 54 59 1 241175 Coding 4 41817 agttctgcctttctaaaggg 53 60 1 241176 Coding 4 41822 agaggagttctgcctttcta 61 61 1 241177 Coding 4 41827 ccggaagaggagttctgcct 57 62 1 241178 Coding 4 42043 ctacactggcttggagctgg 72 63 1 241179 Coding 4 42133 ccaatgctgtctgaactgga 66 64 1 241180 Coding 4 42138 ctgggccaatgctgtctgaa 45 65 1 241181 Coding 4 42143 tacatctgggccaatgctgt 49 66 1 241182 Coding 4 42148 tcagttacatctgggccaat 67 67 1 241183 Coding 4 42410 atcaggattgaaatctactc 74 68 1 241184 Coding 4 43279 atgggagaggagcctggctt 11 69 1 241185 Coding 4 43658 tgagaggatgacatggaaga 45 70 1 241186 Coding 4 43733 ttatctatgacagctgtcaa 19 71 1 241187 Coding 4 44208 tgactcactttcactgtcca 76 72 1 241188 Coding 4 44213 gagcctgactcactttcact 75 73 1 241189 Coding 4 44218 tggaggagcctgactcactt 56 74 1 241190 Coding 4 44223 tgctatggaggagcctgact 54 75 1 241191 Stop 4 44561 taaggttcctaattcccaat 63 76 1 codon 241192 3′UTR 4 44880 tatatgaatatttcccacaa 55 77 1 241193 3′UTR 4 44941 atacatgcactaaaatgagt 67 78 1 241194 3′UTR 4 45105 ctcctaagaaacagacacca 57 79 1 241195 Intron: 4 719 agtcacttaccctcggtttc 6 80 1 exon junction 241196 Intron 4 6356 agagacagggttttaccttg 27 81 1 241197 Intron 4 6818 tgaagaagtttctctctgaa 41 82 1 241198 Intron: 4 20010 cttcagtttgctggagagta 38 83 1 exon junction 241199 Intron 4 25542 ataaagggatggagtcaatg 59 84 1 241200 Intron: 4 28587 aatggaattcctggaaaaac 0 85 1 exon junction 241201 Coding 4 45525 tacactaaaccttactgcat 86 86 1 241202 Coding 4 45779 gagcttgaaattgttttaag 19 87 1 241203 Coding 4 46601 cccactcagaacaaatttta 64 88 1 241204 Coding 4 46996 agataggtagaagttgactt 49 89 1 241205 Intron 4 956 actttaggccaaaagtgccc 27 90 1

[0209] As shown in Table 1, SEQ ID NOs 13, 15, 16, 17, 18, 19, 22, 24, 25, 26, 28, 29, 30, 31, 32, 33, 35, 37, 38, 39, 40, 41, 42, 43, 44, 45, 49, 50, 51, 53, 54, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 70, 72, 73, 74, 75, 76, 77, 78, 79, 82, 84, 86, 88 and 89 demonstrated at least 40% inhibition of human PPAR binding protein expression in this assay and are therefore preferred. More preferred are SEQ ID NOs: 26, 43 and 86. The target regions to which these preferred sequences are complementary are herein referred to as “preferred target segments” and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 2. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 2 is the species in which each of the preferred target segments was found. TABLE 2 Sequence and position of preferred target segments identified in PPAR binding protein. TARGET SEQ ID TARGET REV COMP SEQ ID SITE ID NO SITE SEQUENCE OF SEQ ID ACTIVE IN NO 157644 4 666 tccttttgtggcggatctag 13 H. sapiens 91 157646 4 3904 cggctccatgcaaaatttaa 15 H. sapienS 92 157647 4 3946 accattaagcttgtgcgtca 16 H. sapiens 93 157648 4 8178 catttggtcagctgtttgga 17 H. sapiens 94 157649 4 8183 ggtcaqctgtttggagacat 18 H. sapiens 95 157650 11 386 cagaaqgctctcaaagtaac 19 H. sapiens 96 157653 11 447 gacagaatggactgggctct 22 H. sapiens 97 157655 4 11286 tgttacatcacgtcagatat 24 H. sapiens 98 157656 4 11291 catcacgtcagatatgttct 25 H. sapiens 99 157657 4 11296 cgtcagatatgttctatgtg 26 H. sapiens 100 157659 4 11306 gttctatgtggaagtgcagt 28 H. sapienS 101 157660 4 11331 cctgcaggacagctttgtga 29 H. sapiens 102 157661 4 11355 aaagtggctcaccatgggga 30 H. sapiens 103 157662 4 11360 ggctcaccatggggagaatc 31 H. sapiens 104 157663 4 11365 accatggggagaatcctgtg 32 H. sapiens 105 157664 11 575 ggggagaatcctgtgagctg 33 H. sapiens 106 157666 4 27330 tcagtgacaattgaaggaac 35 H. sapiens 107 157668 4 32297 aagctgtgtcaaaagaacta 37 H. sapiens 108 157669 4 41414 aqacagttgaagacatggtg 38 H. sapiens 109 157670 4 41558 tgagcatgagcatcaaagat 39 H. sapiens 110 157671 4 41622 tcagaacccaattcttacca 40 H. sapiens 111 157672 4 41645 tgttgcaaatcacaqggaac 41 H. sapiens 112 157673 4 41650 caaatcacagggaacggggg 42 H. sapiens 113 157674 4 41666 gggggtctaccattggctcg 43 H. sapiens 114 157675 4 41671 tctaccattggctcgagtcc 44 H. sapiens 115 157676 4 41676 cattggctcgagtccgaccc 45 H. sapiens 116 157680 4 41724 ttcgatggccggcaacacca 49 H. sapiens 117 157681 4 41729 tggccggcaacaccaagaac 50 H. sapiens 118 157682 4 41737 aacaccaagaaccacccgat 51 H. sapiens 119 157684 4 41747 accacccgatgctcatgaac 53 H. sapiens 120 157685 4 41752 ccgatgctcatgaaccttct 54 H. sapiens 121 157687 4 41796 ctcaaccctttatggaagca 56 H. sapiens 122 157688 4 41802 cctttatggaagcagccctt 57 H. sapiens 123 157689 4 41807 atggaagcagccctttagaa 58 H. sapiens 124 157690 4 41812 agcagccctttagaaaggca 59 H. sapiens 125 157691 4 41817 ccctttagaaaggcagaact 60 H. sapiens 126 157692 4 41822 tagaaaggcagaactcctct 61 H. sapiens 127 157693 4 41827 aggcagaactcctcttccgg 62 H. sapiens 128 157694 4 42043 ccagctccaagccagtgtag 63 H. sapiens 129 157695 4 42133 tccagttcagacagcattgg 64 H. sapiens 130 157696 4 42138 ttcagacagcattggcccag 65 H. sapiens 131 157697 4 42143 acagcattggcccagatgta 66 H. sapiens 132 157698 4 42148 attggcccagatgtaactga 67 H. sapiens 133 157699 4 42410 gagtagatttcaatcctgat 68 H. sapiens 134 157701 4 43658 tcttccatgtcatcctctca 70 H. sapiens 135 157703 4 44208 tggacagtgaaagtgagtca 72 H. sapiens 136 157704 4 44213 agtgaaagtgagtcaggctc 73 H. sapiens 137 157705 4 44218 aagtgagtcaggctcctcca 74 H. sapiens 138 157706 4 44223 agtcaggctcctccatagca 75 H. sapiens 139 157707 4 44561 attgggaattaggaacctta 76 H. sapiens 140 157708 4 44880 ttgtgggaaatattcatata 77 H. sapiens 141 157709 4 44941 actcattttagtgcatgtat 78 H. sapiens 142 157710 4 45105 tggtgtctgtttcttaggag 79 H. sapiens 143 157713 4 6818 ttcagagagaaacttcttca 82 H. sapiens 144 157715 4 25542 cattgactccatccctttat 84 H. sapiens 145 157717 4 455251 atgcagtaaggtttagtgta 86 H. sapiens 146 157719 4 466011 taaaatttgttctgagtggg 88 H. sapiens 147 157720 4 46996 aagtcaacttctacctatct 89 H. sapiens 148

[0210] As these “preferred target segments” have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these preferred target segments and consequently inhibit the expression of PPAR binding protein.

[0211] According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other short oligomeric compounds which hybridize to at least a portion of the target nucleic acid.

Example 16

[0212] Western Blot Analysis of PPAR Binding Protein Protein Levels

[0213] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to PPAR binding protein is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 148 <210> SEQ ID NO 1 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 1 tccgtcatcg ctcctcaggg 20 <210> SEQ ID NO 2 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 2 gtgcgcgcga gcccgaaatc 20 <210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 3 atgcattctg cccccaagga 20 <210> SEQ ID NO 4 <211> LENGTH: 48001 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 1662, 1665, 1668, 1671, 12844, 13431-13530, 13749, 15475, 15509, 15576, 20466, 22014, 23090, 23092, 23094, 26394, 30518, 30524, 30673, 36595, 43474, 46942, 47193, 47198 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 4 ccagcctggc caacacggcg aaaccccgtc tctactaaaa atagctgggc gtggtggcgg 60 gtgcctgtaa tcccagctac tcgggaggct gaggcaggag aatcgcttga acccgggagg 120 cggaggttgc agtgagccga catcgtgcca ttgcactcca gcctgggcga caagagcaag 180 actccatctc aaaaaaaaaa aaaaaaaaat taaaaaaaaa agaaaagcta gtctttcagt 240 acagggatct ggagatttta atttgcgagc tgtttaactg ctaaaataaa aaaaaaatgg 300 cggtttccgc aggggagccg aaagctaccg gccggcaggt ttaatatcca gcggcgcgag 360 gcctgagaca gctcagatca aagggaagac tgcgctgaaa gaaatctcgc gaggccgacg 420 cattgaccaa tattcgcgag atttcgatgc cctgtcctct tcctctctct ggcaggacca 480 cggtttatct cgcgaatttt gggaagttcc gttggggaag atggcggcgg cctcgagcac 540 ccttctcttc ttgccgccgg ggacttcaga ttgatccttc ccgggaagag tagggactgc 600 tggtgccctg cgtcccggga tcccgagcca acttgtttcc tccgttagtg gtggggaagg 660 gcttatcctt ttgtggcgga tctagcttct cctcgccttc aggatgaaag ctcaggggga 720 aaccgagggt aagtgactgt aggggaaagg agggggcggg acacaactgg gggagccagt 780 ttgtgagagg aggttcttgc agatgtccca tgggggccac tttaggggca tagagtccag 840 gagagggaat gtgtattagt tttagattgt cactataccc tccttggtga ttatagagct 900 tcgactttag ggcgatgggg actctcaact tctgtgaaga tttcatgctc gactagggca 960 cttttggcct aaagtggtgg gagacggggc aaatgaatac aaaatcagct tacatctcgt 1020 tccagaagag acgcctgaat aaatttaggg ctgtgtgtaa acgtactgga gactattttg 1080 gcatcaatga aatgattagg tccttatatt tattagcagc cagcagttct ctacaatgtc 1140 aagtgttcag atcctgtgcc attgtgtatt tttaaagtgc gctgaatttc gttcttttat 1200 tgaccgaact acgaactgca gaaaaacaac ttttctgtgg aactttacta agattttcta 1260 aagttccaca ctccggctaa gatgctgcct tagcattttg ggcatagttc tgttgataca 1320 gtgaactgtg ttattgttta tcttctccag tttcctctga gctttgcaga ttagagtcct 1380 tgcctgatta tttttgtata gtttctggca cgtaatagtc gctcaaaaag tgttggagca 1440 tcgaagtaca gatatttatc agcaggtttc tcaagaaaaa caaagagtgt gttttggagc 1500 agaggtcgaa aggtgaggcc gggagcagtg actcacacct gtaatcccag cgctttggga 1560 ggctgcggct ggaggatccc tggagcccag gagttcgcga ccagcctggg caacatactg 1620 aggccctgtc tctacaaaaa aaataaaaaa attgccgggc anctngtngg nctcacgcgt 1680 gtaatcccag cactttggga ggttgaggcg ggtggatcac ctgaggttgg gagatcgaga 1740 ccagcctgac caacatggag aaaacccgtc tctactagaa atacaaaatt agccgggcgt 1800 ggtggtgaat gcctgtaatc ccagctacct gggaggctga ggtgggagga ttgcttcaac 1860 ccaggaggtg gaggttgtgg tgagccaaga ctgcgccatt gcattcctac ctgggctaca 1920 agagcgaaac ttcgtctcaa aaaaaaaaaa aaaaaaaaaa ggccgggtgt ggtggctcac 1980 tcctgtaatc ccagcacttt gggaggctga ggtgggcgga tcacctgagg ttaggagttc 2040 aagaccagac tggctaacat ggtgaaaccc tttctctact aaaaatacaa aattagccag 2100 gcacagtggc atgcctgcaa tcccagctac ttggtaggct gaggcaggag aatcacccaa 2160 acccgggagg tggaggttgc agtgagccaa gatagtgcca ctggactcca gtctgggcaa 2220 caagagcaaa actccgtctc aaaaaaaaaa aaaaagaaaa aaaattgaaa ataaataaaa 2280 taaaaaatta gctccaggta gtggctcaag gctgtaatcc cagcaccttg ggaggccgaa 2340 gcgggcgaat cacttgagcc cacaagttgg agaccagcct agacaacctg gcaaaagcct 2400 gtctctacaa cagatacaaa aattagctag gcatggtggc acaagccagt agtcccagct 2460 acttgggagg ctgaggtggg aggattacct gaatcctgta ggtcaaggct ccagtgagcc 2520 agaatcatgc cactgcactg taacctgggc gacagagcga gaccctgttt caagggaaaa 2580 ttagctatag tctcagctac tctgaaggct gagctgggag gatcacttga tcctggagca 2640 tgggaggttg aggctgcaat aggccttgat tgtgccagtg cactcaagct ggggctacag 2700 agtgatactc tgtcttaaaa aaaaaaaaga aaagaaagat gtactccttg ggtgcaagga 2760 gacaacaagt gtaaaaatgg aaacttcatg aagtaggagt ttttgtctag tttgttcatt 2820 gctgtctttt tagcattttc atttatttat ttatttattt gagacagagt tttgctcttg 2880 ttgcccaggt tggagtgcag tggcgcaatc tgggctcact gcaacctccg cctcctgggt 2940 tcaagtgatt ctcctgcctc agccttccga gtagctggga ttacaggcat gtaccaccaa 3000 gcccggctaa ttttgtattt ttagtagaga tggggtttct ccgtgttggt caggctggtc 3060 tcgaattccc aacctcaggt gatccacccg ccttggcctc ccaaagtgct gggattacag 3120 gcgtgagcca ctccgcccag ccagcatttt catttatttt ttttcttttt tttaaatttt 3180 cctctctagc ccagttcaat aacttttttt tttttttttt tgacacaatg tctccctctg 3240 ttgcctagga tggagtaagt ggtgtgatca aggctcatca ctgaagcttc gacctcccag 3300 gctgaggtga tccttctgcc tcagcctcca gagtagctgg ggactgacta caggcacatg 3360 ccaggaggct tggctaattt ttttctttct tttttttttt gagacagtgt ttcgctctgt 3420 tgcccatgct ggagtgcaat agtgtgatct cggctcactg caacctctgc ctcctggatt 3480 caagcaattc tcctccctta gcctcccaag tagctgggat tacaggcatg caccaccaca 3540 tctggctaat tttgtatttt cggtagagac agggtttgac actccagcct ggtcatcaag 3600 agtgaaactt agtctttaaa aaaaaaaaaa aaaaggtgga tagataacag tccagtggta 3660 tggtattcct tacatcagac ccataacttg tgtgtcatct tgccattaaa aatgtcttct 3720 ttggatgttt tgaagtaaaa tgattttgga aaatatttta actgaattct tactgtgatg 3780 aagtgtggga atttattttg caaggaaaac tgtggatggt cttgatagaa cagttactga 3840 taacgttttc tgatttaaat catagagtca gaaaagctga gtaagatgag ttctctcctg 3900 gaacggctcc atgcaaaatt taaccaaaat agaccctgga gtgaaaccat taagcttgtg 3960 cgtcaagtca tggtaaggat tgtggtgaag tgggtaaagt gttttgctta gataattatt 4020 cttaatacgc taagctcccc atgtagatgt gttcatacta tcttcataaa acatctgaat 4080 acatggtggt ttcaacttta ggagtctatt gtatctatac tacaggttga atatccctta 4140 tatgaaatgc ttgggaccgg aagtgtttta gattttggat tgttttggat tttggaatat 4200 ttgcaaatat ataatgagat atcttgggga tgggacccaa gtctaaacac ataatttatt 4260 tatgtttcat aaacacctta tagatatagc ctgaaggtaa ttttatacaa catatttaat 4320 aatatattta atacttttgt gtgtgaaact aagttttttt ttgttttttt tttttattga 4380 gacggagttt cactcttgtt gcccaggctg ggttgtaata gcccgatctc ggctcactgc 4440 aacctctgcc tcccaggttc aagtgattct tctgcctcag cctcccgaat agctgggatt 4500 acaggcatgc gccaccacgc ccagctaatt tttatattat ttgtagagac ggggtttctc 4560 catgttggtc aggctggtct tgacctccag acctcaggtg atccacctgc cttcgcctcc 4620 caaagtgctg ggattacagg cgtgagccac cgcgtccagc cgttttgact gcattttgac 4680 tatgaccctg ttatgtgagg tcaggtgtga aatccttggt ggcatcattg ggaatgctaa 4740 aaaggttttg gatttgggag catttcagat ttttgatttt cagattaggt atgcccagcc 4800 tgtactagta gaatttttga ttcattcttt tttttgagac caggtcttat catccaggct 4860 caagtgcagt ggcgcgatca tggctcactg cagcttcaac ttcccaggct caggtggttc 4920 tcccacctca gcctcctagt atctggaaca acaggtgcat gtaaccatgt ccagctagtt 4980 tttttttttt tttctttgag atggagtctt gctctgtggc caggctggag tgcaatggtg 5040 cgatctcggc tcactgcaac ctctgcctct taggttcaag agattcccct gcgtcagcct 5100 cccgagtagc cgggactaca ggcacacgcc accacgcctg gctaattttt tgtattttag 5160 tagagatggg gtttcaccat gttggtcagg atggtctcca tctcctgacc tcgtgatctg 5220 cctcccaaag tgctgggatt acaggcgtga gccactgtgc ccggctgccc agctagtttt 5280 ttatattttt tgtagaaatc gttttttttt tttttttttt gagatggagt gtcgctttgt 5340 cacccaggct ggagtgcagt ggtgcgatct cggctcactg caagctttgc ctcccgggtt 5400 catgccgttc tcctgcctca gcctcccgag tagctgggac tacaggcccc tgccactatt 5460 cccggctaac tttttgtatt tttagtagag acggggtttc accgtgttag ccaggatggt 5520 ctcgatctcc tgacctcgtg atccacccgt ctcggcctct caaagtgctg ggattacagg 5580 cgtagccact gcgcttagcc agaaataggt ttttactctg tggctcaggc tggtctcaaa 5640 cttgtgggct caagtgatcc acccactttg gcctcccaaa gtgttaggat tacagtgtga 5700 gccaccgtgc ctggcggatt catttttttt tttttttttt aaagatggaa tctctgtctg 5760 tcacctaggc tggagtgcag tggtgggatc ttggctcact gcatcctccg cctcctgggt 5820 tcaagcgatt ctcctgcccc agcctcctga gtagctggta ttacaggcat ccgccaccat 5880 gcccagttaa tttttgtatt tttagtagag atggggtttc accatgttag ccaggctggt 5940 cttgaactcc tgaccttgtg atccacctgc ctcagcctcc caaagtgctg tgattacggg 6000 tgtgagcccc tgtgcctggc ccaggttgtc atttttattc cttaaggcaa ccaaattcta 6060 gcttgaaata tgttctgtaa atgatactgt cattagagag acattgtgag atagaaggat 6120 tccaggtggc taataaggcc ctctctttgg gtaccactct cttggtttgg tcaccatgac 6180 tttgcttgtc tacttttcag ttttgcagat tcagaaacct aattttattg gtacatcaag 6240 attttataga gttttcatgg ctgggcgtgg tggttcacat ctgtaatccc agcactttgg 6300 gaggctgagg cgggtcgatc acctgaggtc aggagttcga gaccaggctg accaacaagg 6360 taaaaccctg tctctactaa aaagacaaaa ttagctgggc atagtggcag gcgcctgtag 6420 tcccagctac tcgggaggct gagacagcag aaccgcttga acccgggagg tggaggttgc 6480 agtgagccaa gatcatgcta ctgcactcca gtctgggcat cggagcgaga ctccatctca 6540 aaaaaaaaaa aagagttttc aacaactgac atactgtatg cttatttatt aatttatgac 6600 aaaacattta gttttccagt tttcataata ctggtattca ttgaccagag cacctcttct 6660 cctgtcaaga atcaagagat ttcagattat taataaatta ctttgttttg taagggagaa 6720 ttagagcagt gagatagatt tatttgcaac atggaaaagc ctggaaaact atccttatac 6780 cagaaaactg ttcacgtttt tagggactgg aatgtttttc agagagaaac ttcttcactg 6840 gatagggtta ggaaaactag gctaagtttt tttttgtttg tttgtttgtt ttgttttgtt 6900 tgagatggag tctcactctg tcaccaaggt tggagtgcaa tggtgtggtc ttggctcact 6960 gcaacctctg catccccggg tcaagcgatt ctcctgcctc agcctcctga gtagctggga 7020 ctacaggcgc gtaccaccac acctggctaa tttttgtatt tttagtagag atggggtttc 7080 actatgttgg ccaggctggt ctcgaactcc tgacctcatg atctgctcgc cttggcctcc 7140 caaagtgctg ggattacagg cgtgagccac tgcaaccggc ctcaaactag gctaagtttt 7200 tgttctttgg aagtcccttt tctggactac atgttctgtt tcttttcttt ttcttttttt 7260 ttttgagaca gagtctctct ctgtcgccag gctggagtgc agtggtgtga tctcggctca 7320 ctgcaacctc cgcctcctgg gttcaagtga ttctcctgcc tcagcctgct gagtagctgg 7380 gactataggc gtgcaccacc acgcccagat aatttttgta tttttagtag agacggggtt 7440 tcaccatttt ggccaggatg gtctcagtct cttgacctcg tgatccaccc gccttgacct 7500 cccaaagtgc tgggattaca ggtgtgagcc accgcaccca gcctatatgt tctgtttttt 7560 agagtcaatc tcattttctt aaaatttggt tgtgtttctc agattacatg caaatcacct 7620 tgttaaattt gtacactacc aaatctcctg agccatctta tttaactgtt tatatcttag 7680 catgtggtca cttccacagg cggcagatct catgtattac attttgattc ttcctaatat 7740 ttctctaaag gtttttttgt tttagatgtg gaaatacaac gttttctttt ttgtttttga 7800 ctaaatagaa acaggatctc tctatcttgc cttggctggt ctcgaactcc tggactcaag 7860 tgattattca gctttggcct ctcagtgtgc tagaattaca ggcatgagcc acgtgcctgg 7920 ctgaggaata cctttttcta cactcatata tatggatttt gcatggatgg acagagtaac 7980 acttgtagac cagtgttact ctatatagtc tacaaaatct atagactata ttgagtctat 8040 agactgtcct gcaatattac tactaggata atgtgtatgt ttacctaaaa cactgttggc 8100 ctggttaatt ttcacctcaa atgttttccc acttgacaca ggagaagagg gttgtgatga 8160 gttctggagg gcatcaacat ttggtcagct gtttggagac attgcagaag gctctcaaag 8220 gtttgtgaac acttttgaaa tatgctgatt tcacaaaacc cccagttcaa gaaggaccat 8280 tttatactta aactagaagt aaaaaattga ggtaggtcaa ctttatgtat tagatattgc 8340 tcttgcattc atttatggag ttggaatgat ctttttgcta aagtttgaaa catttatttt 8400 tgatcctgtt ttggaggctg atagagattt aaaagataaa tgatgaaggg taatataaga 8460 tacctgggga ggctgggtgc ggtggctcgc tcctgtaatc ccagcacttt gggaggctga 8520 ggtgggcgga tcacaaggtc aggagtttga gaccagcctg gccaacatgg tgaaaccctg 8580 tctctactaa aaatacaaaa attagctgga cgtggtggcg ggcacctata atcccagctt 8640 ctcgggaggc tgaggcagga gaacagtgtg acctggggag gcggaggttg cagtgagcca 8700 aaatcacgcc attacactcc agcctgggca ggagggtgag tcttgtctca aagcaaaaaa 8760 aaaaaaaaaa aaagatacct gggaaaagtt ttttttccct tcacatttat ttttgattaa 8820 tctcgataag aagatggact tttttttttt tttttttttt ttttgagatg gagtttcact 8880 cttgttgctc tggctggagt gcagtggtgg aatctcggct caccacaacc tctgcctcct 8940 gggttcaagc gattctcctg cctcagcctc ccaagtagct gggattacag gcatgtgcca 9000 ccatgcccgg gtaattttat atttttagta gagacagggt tttccatgtt ggtcaggctg 9060 gtctcgatct cttgacctca ggtgatctgc ccaccttggc ctcccaaagt tctgggatca 9120 caggcgcgag ccacagcaac tcgcgaagtt tttttttttt tttttttttt tccgagacgg 9180 tgtctcactc tgtcacccag gctggagtgc agtggcacaa tcttggctca ctgcaacctc 9240 cacctcctgg gttcaagcga ttctcctggc tcagcctccc aagcagctgg gactacaggc 9300 gcgtcccacc acgcctggct aatgttttgt atttttagta gagatggggt ttcaccgtgt 9360 tagccaggat agtatagatc tcctgacctc gtgatccacc cgcctcggcc tcccaaattg 9420 ctgggattac aggcgtgagc taccatgcct gtccagaaga tggacatttt aactctaaaa 9480 agatttttac ggtgtttatg gagcaaattt ccaggaaagc aaatgtaaaa tataagtcat 9540 gagcttgatt attcatgctt ataatgacta attctatttt tggggaataa tgaaagtaaa 9600 aacagatttt actgtgatcc accagaaatg aacctaattc agctgagggt tttttttttt 9660 ttttctggag acagtgtctc actctgtcgt ccaggctgga gtgcagtggc acgatctctg 9720 ctcactgcaa cctccgtttc ccggattcaa gtgattgttc tgcctcagcc tcctgagtaa 9780 ctgggattac aggcgcccat taccatgctc agctaatttt tggattttta gtagagatgg 9840 ggtttcacca tgttggccag ggtggtcttg gactcctgac ctcaggtgat ctgcctgcct 9900 tggcctccca aagtgttggg attacaggcg tgagccactt tgcccagtct cagctgagta 9960 ttatcttctt tacctttttt tgttttcttt ttctttgaga cagagtgtcg ctgtgtcacc 10020 caggctggag tgcagtggca cgatctctgc tcactgcaac cactgcgccc ccaccgggtt 10080 caagagattc tcttgcctca gcctcccgag agggtgggat tataggcgcg caccaccaca 10140 cctggctaat ttttgtattt ttattagaga cagggtttca ccacgttggt caggctggtc 10200 ttgaacccct gaccttgtga tccgcatgcc tcggtctccg aaagtgttag gattacaggc 10260 gtcagccacc gcacgaggcc tttttttctg ttttaattgt ggtaaaatat aggtaacaaa 10320 atttaccatt ttctttgttt ctaagtgtat agttcagtag cgttaagtac attcatatta 10380 ttgtataatc atcaccactg tccatctgta aaacttcact catcttccca agcttacatt 10440 ctgtaatgtc ccattcccgc cttcccccag tccctggcag ccaccattct gcttcctgtc 10500 tttatgaatt tgactactct gtatacctga tataagtgga atcatatatt tgtccttttg 10560 tgactggctt atttcactaa acgtaatgtc ttcaaggttc atctattttg aagcatttat 10620 tagaatttct ttctttttta aggttgaaaa atactcctct ttatgtatat acatttcatt 10680 tatccattca tctgctagtg gacacttgga ttgttttaac cttttgactg ttgtgattaa 10740 tggtgttgtt aatgttgagt tttttttaat gttagtttta atgttaatct tccttattct 10800 atcattagca gataaatgtg tcttaagtta aaggcagttt gtcccacata aggagtaatg 10860 aaaacaggag aggagaaagg ttcggtaatt tttcaaatag atcttttttt tttgaccttt 10920 agtactgcat aggccacagc taaccctaat cagtgaactt tatgtaaatt acaagcattt 10980 gaactactga atatgtttac ggttttggaa ggatgaatct ttctgtctta aaagaatatc 11040 atttaagtca tttggagcat tctttgtgac tccttttttc atccttttat agtaacatct 11100 ttaccagcaa tgactgatcg tttggagtcc atagcaagac agaatgggta agtagtcttt 11160 ctgtaggcag aaatgtttaa cttagtgtta actttgggtg gctttattta tgtgtcttct 11220 attaattttt gttttgattt gttcctgcta gactgggctc tcatctcagt gccagtggca 11280 ctgaatgtta catcacgtca gatatgttct atgtggaagt gcagttagat cctgcaggac 11340 agctttgtga tgtaaaagtg gctcaccatg gggagaatcc tgtggtatgt gttgttgata 11400 tttcactgga atctgtgggg tttacttagg gacattaatt ttaaaattaa ttgttggggt 11460 ttaaatagct tgggtttagg tttttccatg agccactgta gagatgatgt gatgggctac 11520 aaaatcaaca gctatagaat gggatttaga aaatatgggt cccatctctg acatcatcca 11580 aaggctttaa caagtggaaa caccatagga acccttttcc ctttccttcc ttccttcctt 11640 cctttctttc ttttctttct tttttctttc tttctttctt gcttgcttgc ttgctttctt 11700 tttcttttct ttcttttctt tctttctctt tctctctttc tttctttctt tccttccttc 11760 cttccttcct tccttccttc cttctttctt tctttctttc tttccttctt tccttctttt 11820 tctttttttt tttccctggg acagagtctc gttctgtcgc ctggagtgca gtggcatgat 11880 cttggctcac tgtaatctct gcctcctgga ttcaagcaat tctcctgcct cagcctccca 11940 agtagctggg attacaggtg tgtgccacca tgcctggcta atttttgtat ttttagtaga 12000 gacggggttt ccctatgttg gccaggctgg tctccagctc ctgacctcaa gtgatccacc 12060 tgcctcagcc tcccaaagtg ttgggattac aggcataagc cactgcaccg gcaggaaccc 12120 ttttcatttc atatcaacaa acatgagcca cgattgaaca ataggtgctg gggatacaga 12180 aataaatgac tcagggttac taccttcaaa gagattataa tcaagaacag atgatctgaa 12240 aaattatgta atagagatga atgtaaaatc tgtgtaacca gataatttga tatgaattcc 12300 atgtatggca ttagagttcc taatgtttta aacaagctac tgaaagtaga ttcttttaaa 12360 aactatttca tatgctttga ctttaaattt ctcaccctaa tcctgtggtt caccaagtag 12420 gaagaagatg ctttttaata ttttaatatg aggaccatct gtagttttaa tggttttgtt 12480 gtaaaacatg tgctatagaa aaagagaaag gcttttgata tcttcaaagt tgctaaagta 12540 gttttaatgt tttaacataa ttttctttga tttcagagct gtccggagct tgtacagcag 12600 ctaaggtgag caaatacgat ttatattcct tgatattgac agacagtgct gttctttaca 12660 gctggctttt aagactggtc agaagttatt ctcctcagga acaagtttat tttcttgggc 12720 aaagattttt aagagtctat acttaatcac gtcctgaatt gacatagcat agcttttcac 12780 cctactctat cccagggttt ttttgttttc gtttttgttt tgagatggag tcttgctcta 12840 ttgncccagg ctggagtgca gtggcgcgat ctcagctcac tgcaacctct gcctcctggg 12900 ttcaagcgat tttcctgcct cagcctccag agcggctggg actgcatgtg tgtgccacca 12960 tgcccagcta atttttgtat ttttaataga gactaggtgg ctgggtgcgg tggctcacgc 13020 ctgtaatccc agcactttgg gaggctgagg caggtggatc catgaggtca ggagttcaag 13080 accagactgg ccaagatggt gaaaccccat ctctattaaa aatataaaat ttaggctgac 13140 acctgtaatc ccaggacttt gggaggctga ggcgggtgga tcatgaggtc aggagatcaa 13200 gaccatcctg gctaacacgg tgaaacccca tctctactaa aaaaagaaaa aatacaaaaa 13260 attaactggg tgtggtggca ggcatctgtg gttccggctg cttgggaggc ttggtctgga 13320 gattgggtta agggttggag cggaccttgc atataggtga aatgtcgccc ctgcactccc 13380 agccggggcg acagagcatg actccctcca aaaaccaaat tgaacacaac nnnnnnnnnn 13440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn tttttgtgtg gctggaactt tgattggtgt 13560 aatcagaaag ctgacccagg agaaggcgtg aacctgggaa cggagcttcc agtaagctga 13620 gatctcccca ctgcactcca gcgtgggcga cagagcaaga ccccgtctca aaaaaaaaaa 13680 aaaaaaaaaa aaattagcca ggcatggtgg tgggtacctg taatcccagc tactcgggag 13740 gctgaggcna gaaaattgct tgaacccagg aggcggaggt tgtagtgtgc tgagatcgtg 13800 ccactgcact ccagcctggg tgacagagca agactccatc tctagaaaaa aaaaaaaaat 13860 agagacgagg tttcgccatg tgggccaggc tgatcttgaa ctcctgacct cacatctccc 13920 aaagtgctgg gattacagtt gtgagccacc atgctggacc cttttctttt cttttctgtt 13980 tttttttgag acggagtttt gttcttgtta cctagtctgg agtataatgg tgcggtctcg 14040 gctcactgag cacctggccc cttttctgtt tttttatttt tatttttatt tttttggtga 14100 gacggagatt tacttttgtt gcccaggctg gagtgcaatg gtgtgatctt ggctcaccgc 14160 atcctctgcc tcctgggttc aagtgattct cctgcctcag cctcccgagt agctgggatt 14220 acaggcatgt gctaccacgc cctgctaatt ttgtattttc agtagagacg gggtttcacc 14280 gtgttgccca ggctgatctc aaactcctga cctcaggtga tcctcctgcc ttggcctccc 14340 aaagtgctgg gattataggc atgagccact gtgcccggcc cctgtttttt taagttttaa 14400 tttttttttt cttattttga ttcagattcc caacagaaag atatcccagg gtctcttaaa 14460 cttggtacta atgacaattt ggattggaaa attctttgct cccgggtggc ctggtgcatt 14520 ctattatgtt taatagcatc cctggcctct tcattagttg ctagtagtat ctccttagtt 14580 gttacaacca tgtcccctgg ggcacaaaat cacccctggt tgggaattgg tgatctaggt 14640 ttaggaattt catatagctg ggggcggtgg ctcatgccta taatcccaac aatttgggag 14700 gtcgaagtga gcagatcact tgaggccaag agttcaagac cagcctagcc agcctggcga 14760 aaccctgtct ctacaaaaaa tacaaaaatt gctgaatgtg aggccaggcg cgatggctca 14820 tgcctgtaat cccagcactt tgggaagccg aggcaggcgg atcacctgaa gtcaggagtt 14880 cgagaccagc ctgaccaaca cggagaaacc ccgtctctac taaaaataca aaattagccg 14940 tgcgtggtgg cacatgcttg taatctcagc tacttgggag gctgaggcaa gagaatcgct 15000 tgaacccggg aggcagaggt tgtggtgagc caagatcatg ccattacagt ccagcctggg 15060 caacaagaac aaaactctgt ctcaaaaaaa aaaaaaaagt agctgagcat agtggctcac 15120 acgtgtaatc ccagctactc aggaggctga ggcacaagat ggcttgagcc tgggaggtgg 15180 aggttgcagt gagctgggat cacatcactg cattccagcc tgggtgacgg agtgagactg 15240 tctcaaaaaa aaaaataata aagaattatg tgtggaacaa aataaataga aggtaatcat 15300 tgaatcagtg aaggcttgat aattgagtat gataccatga aaataacttt ttcgctttga 15360 ttttgggaaa aattgtttca tttagaaggt acatgtgtgt tacagtaaaa gtgctaggct 15420 tgaagacagt cgcttttagc agatagtgga ttttttcact tttcttttaa ttgangtgga 15480 aatttctgta cttaaattta aactcagang ttaagacttt tggggaattt aaggaagtta 15540 ttacatacac tttgttttga gcattgcgaa gttctncagg caccacatga ggatgtctta 15600 gagtgttcac attgaggaaa taagttgtgt atgtgtgtgt gcaaaagctt tttttttttt 15660 ttttttcttt tttttgagtt ggagtcttgc tctgtcgccc aggctggagt gcagtggtgt 15720 gctctctgct cactgcaagc tccgcctcct gggttcatgc cattctcctg cctcagcctc 15780 ccgagtagct gggactacag gcacctgcca ccatgcccgg ataatttttg tatttttagt 15840 agagacgagg tttcatcgtg ttagccagga tggtttcgat ctcctgacct cgtgatctgc 15900 ccgcctcggc ctcccaaagt gctgggatta caggtgtgag ccaccgcacc cggccttttt 15960 tttcgagatg gtgtgtcact ctgttgctca ggctggagta cagtggcgca atctcggctc 16020 actgcaacct ccccctcccg ggttcaagcg attttcctgc cttagcctcc tgagtagctg 16080 ggattatggg tacctgccat tatgcccggc taatttttgt agtatccagg catggtgaaa 16140 ccttatgtct actaaaaata caaaaaatta gccgggtgtg gtggtgctcg tctgtaattc 16200 cagctacaca ggaggctgag gctggagaat tgcgtgaact caggaggtgg aagttgtagt 16260 gagctgagat cgtgccatcg ccatcggact ccagcctgga cgacagagtg agactctgtc 16320 tcaaaaaaga aaaggagaaa aaaagaaata atgttgactt ttacctgata ttaccattta 16380 gtatagatgg taatattaag ttaaagaata tattttgctg ctactcgttt ctttttgctg 16440 tcttaatagc actgttgact gtaagtgctc agtaaatgct cacagaatgt ctgctcaagt 16500 gtctgacatt gtcatggcag tttagctttc cagagagctt tttttttcaa gtttgaaaaa 16560 agatatcaag agttagaatt gtggagatgt attggattcc taaacctatc cacaaaagcc 16620 catttgtccg acaatcgaag cctgaatatt gggtcctaga aagccagagg tcacggtagt 16680 agtttatgtt ttctagggta gatctggtcg tagcaatatt ttctaatggg tattttgcct 16740 cgagagtacc cactaccttt tctctgggtc tccatctctg tcctagtatt tgttttccct 16800 ctcatctttg ctctcatgtt gctgtatacc cacaagtgtt gtgtcttccc aatttatttt 16860 ctttttttga cacacagtct tgctctgttg cccaggctgg agtgcagtgg cgcgatctcg 16920 gctcactgca agctccgcct tcttggttca tgctgttctc ctgcctcagc ctcctgagta 16980 gctgggacta caggtgcccg ccaccacact cggctaattt ttttttgtat ttttaataga 17040 gatggggttt caccatgcta gccaggatgg tctcgatctc ctaacctcat gatccaccca 17100 ccttggcctc ccaaagtgct gggattacag gcgcgaggac tgtgcctggc ccttcctaat 17160 ttattttctt cttattatct ctgcaatcaa aagttttatt tttgttgaca tccaaattca 17220 gttacatatt tggaggaaag gagctgggtg tggtggtgca cacctgttgt tgaaggtgag 17280 aggttgaagt gagagcattg ctgaagtcct ggagttagag tctggcctgg gcaacatcgg 17340 gaaaccccgt ctctacaaaa aaattaataa aaagggaaaa gaaaggaaag ttgttaatga 17400 ttgatttaaa tcaggtttca ttgcttggga gctaataaaa cacctggtaa agagattttg 17460 cccaccttcc ttttaacttt cacactgctt attagcacct tctccagaaa aaatggagga 17520 gggagacaaa ggaaaatatt ttcttttctt ttgctgcttt acattgatgc tagaaaactt 17580 ttgaggaaaa tggttaaagc tgattattaa aatcagtttt atatagctgt gatttttggc 17640 actttgttgt aagattgagt agtaatttta tgttttattt tcctctcttt cccctcttcc 17700 ctgaactcct tatagggaaa aaaattttga tgaattttct aagcacctta agggccttgt 17760 taatctgtat aaccttccag gggacaagta agtattttta atatttttgc ttttgtttgt 17820 aaaaatcagt ataaatctat aagccacctt atgcctagaa atttgccacc tcttctgctg 17880 tttgtcactc ctagtttcta cttatactgc aatctgtagc tcagcttcca acattgggcc 17940 tttcaaagat atcttcaata ggcagcacct gcattaatcg atcataatgc tggaagacag 18000 aatataatct ttgagatgtc caacattcca atttctaggc tgaaaggaat tagttgctgt 18060 ggaactggct ttaattctat ttttaaccta cctgggattc tattaacgta tttggctttt 18120 taaaacagag ataaaaataa gctttgaagt gctaaaaata ggctttaata gtagtgaagc 18180 cagtcaaagt ggcccatgcc tctaatccca gtacattggg aggccaagac aggaagattt 18240 cttgagctca ggagatcaat accagcctgg gcaacatagt gagaccctgt ctctagcaga 18300 aagacaacag caacagctta gatgggtggt gatgggtgca tgtagtccca gctacttcgg 18360 aggctaaggt gggacgatca cttcaaacta gtagtttgag ggtgcagtga gcagtgattg 18420 cgtcactgtg ctccagcctg gttgacagag cgagatgctg tctcaaaaaa aaagaaaaaa 18480 tatgtgtgta tatatatata tatatatagt aacaaaaaaa atttatttta aaaattaatg 18540 ttttaggctg ggcacagtgg ctcactcctt taatcctggc cactttggga ggctgaggtg 18600 ggtggaccac ctgaggtcca gtcaagaatt aatgttttaa aatgtttttt ttttttcttt 18660 ttttttaaat acggattgtc gctctgtcac ccaggctgga gtgcagtggc atgacctcag 18720 ctcactgcag cctccacctc ccgggttcaa gcagttctcc tgcctcagcc ttccgagtag 18780 ctgggtttac ggctatgcgc caccacgccc ggctaattat tataattttt tttgagatgg 18840 actcttgctc tgtcgcccag gctgcagtac agtggtgcca tctcggctca ctgcaacctc 18900 cgcctcctgg gttcaagcga ttctcctgcc tcagcctccc gagtagctgg gattaaaggc 18960 gcccaccact gacctggcta agttttgcat ttttagtaga ggtggggttt caccatcttg 19020 gccaggctgg tctcgaactc ctgaccttga tccacccgcc atggcctccc agagtgcttg 19080 gattataggc gtgagacatt gcgcccagcc acctggctaa tttttgtatt tctagtagag 19140 acagggtttt accatgttgg tcaggctggt ctcaaactcc tgacctcacg tgatctaccc 19200 gcttcggcct cccaaagtgt tgggattaca ggcgtgagcc accgcgcctg gccagttttt 19260 tttttggttt ttgttttttt ttggagacag agtctcgctc tgccaccagg ctggaatgca 19320 atggcatgat ctcggttcac tgcaacctcc acctcccggg ttcaagcaat tctcttgcct 19380 tagtctccca agtagctggg actacaggcg catgctgcca cgcctggcta attttttttt 19440 tttgtatttt attagagacg gggtttcacc gtgttgccca ggctggtctc aaactcctga 19500 gctcaggcaa tctgccctcc tcggcttccc aaattgctag gattataggc atgagccact 19560 gcgcctggcc agtttttaaa aatatatata tatttttgcc ttcaatttag acttgctctt 19620 taggccttct atgtcactgt ttgtgtctgt atgtatatat gtgtattttt caactttttt 19680 tttttttttt tttgagatgg aatcttgttc tgtcacccag gctggagtgc agtggtatga 19740 tcttggctca ctgcaacctc ttcctctcgg gttcaagcaa ttctcctgcc tcacccttcc 19800 gagcagctgg gattacaggt gcctgccacc acgctcggct aatttttgta gttttagtag 19860 agatggggtt ttgccatgtt ggccaggccg gtctcaaact cctgacctca ggtgatctgc 19920 ccgcctcagc ctcccaaggt gctgggatta caggctgagc caccccgctt ggtcatattt 19980 ttcaactatt aactcatgtt cttctcatct actctccagc aaactgaaga ctaaaatgta 20040 cttggctctc caatccttag aacaagatct ttctaaaatg gcaattatgt actggtaaga 20100 ctgctaaaac cttaattcta catgatccct tgagtttttc tctggggaga ttagggtcct 20160 attttataca tctctattaa gcctgtatga taatatgtcc tttctcttcc aaaatgatga 20220 ttgtatttga acttctgagt atcagtatcg ctaagttgtt ggttaccata ggttttatct 20280 caagactcat cagacctgga gtcccatctg agaaataata gtctaagtgt aatttttgtt 20340 caccatacta catactggtg tgtacttgga aataatccct ttactttgga ggtgaaatac 20400 atttgctata tgcccaatta caagacagaa ccattatgat agtattataa gactttgggc 20460 tgggcngcgg tggctcacgc ctgtaatccc agcactttgg gaggccgagg cgggcggatc 20520 acctgaggtc acgagttcga gaccagcctg cccaatatgg tgaaaccccg tctctactaa 20580 aaatacaaaa aattatctgg gcgtggtggc gggcgcctgt aatcccagct actcgggagg 20640 ctgaggcagg agaatcactg gaacctggga agcggaggtt gcggtgagct gagattgtac 20700 cactgcactt cagcctgggc gacaagagcg aaactccgtc tcaaaaaaaa aaaaagacaa 20760 gacttcggag aggtgaactc aatatcacag tgtgtaccaa gaccatcagg gattaaatat 20820 aagcaaacat agatcacttg tcttacagga aagcaactaa tgctggtccc ttggataaga 20880 ttcttcatgg aagtgttggc tatctcacac caaggagtgg gggtacgtga tcctattttg 20940 cttgtttaga aacatgtgta atttaagttg agggtcatta cttttcattt cacctttagt 21000 ttgtttgttt atttttttga gacagagtct cactttgttg cccaggctgg agtgcagtgg 21060 catgatctcg gcttactgct ccttccacct cctgggctca agtgaatctc atgcctcagc 21120 ctcctgagta gctgggatta caggtgtgca ccacaatagc cagctaattt attctattgt 21180 tggtagagac agggtttcac tgttttggcc aggctggtcc tgaactcctc gcctcgactg 21240 atcggcctac cttggtctcc caaagtgctg ggattacagg catgagccac tgcgcccagc 21300 ctatttattt gttaattttt attctgtttt tttttttttt ttttaagacg gagtcttgct 21360 ctgttgccca ggctggagta cagtggcgcg atctcagctc actgcaagct ccgcctccca 21420 ggttcacgcc attctcctgc ctcagcctcc agggtagctg ggactacagg cgcccgccac 21480 cacgcctggc taattttttt gtatttttta gtggagacgg ggtttcacag cgttagccag 21540 gatgatctcg atctcctgac ctcgtgatct gcccgcctca gcctcccaaa gtgctgggat 21600 tacaggcatg agccactgcg cctggccaat ttttattctt atgtatttgt ttatttttct 21660 tgagacaggc tctcaccagg ctggagtgca gtggcacaat cttggatcac tgtaacctct 21720 gcctcctggg ttcaagggat tctcctgcct cagcctcccg agtagctggg attacaggca 21780 tgtgccacca cacccgacta atttttgtat ttttagtaga gatggggttt caccatgttg 21840 gccaggctgg tctcaaattc ctgacttcaa gtgatcctcc ctcctcggct gcccaaagtg 21900 ctgggattac aaacaggtat tagccactgc accaatccct atttatttat ttttgagaca 21960 gggtcttgct gtgttgccca ggctggagtg cagtcgtgca atcatgactc actnccagcc 22020 tcaacctcct gggcccaaat aatcccccta actcagtttc ctgagtagct tggactacag 22080 gtacaggata ccatgcctgg ctaatttttg ttgttttata gagacatggt cttactatgt 22140 tgcctaggct ggtcttgaac tgggatcaag tgatcttcct gccactgcat tgcaaagggt 22200 tgggattata agcataagcc actgcgcccg gcctcatctt ggggtttata aacatataag 22260 cagagggaga aaattgccag atatttgtgt attttgtgat gatttcttcc tttttttttt 22320 tttttttttt tttttaggca tttaggcaga gtcttgctct gtctcccagg ctggagtgca 22380 gtggcgcgat cttggctcat tgcaacctct gcatcctggg ttccagcaat tctcttgcct 22440 cagcctccca tgtagctggg attacaggca cgtgccaccg tgcccagcta atttttgtat 22500 ttttaccaga gacagggttt caccatgttg gccaggctgg tctcgaactc ctgaccctag 22560 gtgagctgcc tgtctcggcc tcccaaagtg ctgggattat aggcgtgagc cactgcgcct 22620 gggctttttg cgatgatttc taagctgtca atacctgtcc tccccatttt tgtttgttat 22680 ctttttgaga cagagccttg ctatgtccct ctggctgaag tacagtggca gaaacatagc 22740 tcacttcagc ccttaactct tgggcttaag tgatcttccc acctcagccc cctgagtagc 22800 taggacttca gtcaagtgcc accatgcctg ggtaacttta acaatttttt tagagatggg 22860 ggtctcactg tgttgcctag gctagagtgc agtggtgcag tcataactca ctatagactt 22920 gaactcctgg cttcaagtga tcctccagcc tcagcctccc aaagtgttgg gattataagc 22980 gtgagccagt tcacctggcc tttttttttt tttaaatcaa ttgtgtgaaa taaattgtta 23040 gtcccggaat ccatacttca tcttctgtca tatgtctttt ttttttgagn antnggagtc 23100 ttgctctgtc acccaggctg gagtgcagtg gcacgatttt agctcactgc aacctccacc 23160 tcctgggttc aagcaattct cctgtcacag cctcccgagt agttgggact acaggtgcga 23220 gccaccatgc ctggctaatt ttttgtattt ttagtagata caaggtttca ccatgctggc 23280 caggctcgtc ttgatctcct gacctcgtga tctgcccacc ttggcctccc aaagtgctgg 23340 gattacaggt gtgtgccacc acacccaagc gcctgtcata tttctttatt ttttattttt 23400 actttttgag acggggtctt gctctgcctt ccaggctgga gtgcagtggc acaatcacgg 23460 ctcattgtat ccttgacctc ccaccttaac ttcctgagta gctgggacta cgggcactca 23520 ccagcatgcc aggctaatta ttattattat tatttttgag acggagtctc cctctgttgc 23580 cagggctgga gtgcagtggc atgatctcag ctcactgtaa cctccgcttc ctgggttcaa 23640 gcgattcttc tgcctcagcc tcctgcatag ctgggactac aggcgcgtgc caccatgccc 23700 agctagtttt ttgtattttt agtcaagatg gggtttcacc atgttagtca ggatggtctc 23760 aatatcctga ccttgtgatc cgcctgcctt ggcctcccaa agttgtggga ttacaggcat 23820 gagccaccgc acctggccaa ttttttgtat ttttagtaga gatggggttt caccatgttg 23880 gccaggctgg tcttgaactt ctctcctcag gtgattcacc atctttacct cccaaagtgc 23940 taggattaca ggcattagcc atcgcgcccg gcctattatt attattttta gaggtggagt 24000 ctgactgtgt tgaccatgct agtctcaaac tcctggactc aagtgatcct cttgccggag 24060 cctcccagag agccaggatt acaggtgtga gtcaccacac ctgccatatt tcataagctg 24120 ttttggagca tgactcttca tatatgttaa agtactttct tgaatatcgg tttatataat 24180 aaatgttaat gtttttctgt gtttgtaatt tacctgataa agatacagtt atgagtttta 24240 ttttttatag gtcatttaat gaacctgaag tactatgtct ctccttctga cctactggat 24300 gacaagactg catctcccat cattttgcat gagaataatg gtaagacttt aattgtaaag 24360 gaattaaagg ttttgttcgg tgcccagctt gcctttaatt tctggtttgc tattctctaa 24420 tgagtttgtt gaccttacca tttgcacctt atacacacaa cacctcacac acgtatatgt 24480 gtgttcacaa gtacatacat atgtatatac acacatgcat atatacacac atacataata 24540 ggagcttaat aagtactttt gggctgggca cggtggctca cgcctgtaat cccagcactt 24600 tgggaggctg aggtgggcag atcacgaggt caggacttgg agaccagcct gaccaacatg 24660 gtgaaaccct gtctctacta aaaatacaaa aattacccaa gcatggtggt acgcgcctgt 24720 aatcccagct actcaggagg ctaaggcaag agaattgctt gaacccggga ggaggaggtt 24780 gcagtgagtc aagattgcgt cactgcactc cagtctgggt gacagagcga gactccatct 24840 caaaaaaaga aaaaaagtac ttttgaataa ataagtaaat gagcaactca taagaagaga 24900 tataaacgat ctgacctgag ataatcagct tactcattgt ttaaaaagtg tagaaggatg 24960 ggcctggtag tttatgcctg tagtcctagc tacttgggag gtttgggcgg gaggattgct 25020 tgagcccagg agtttgagac tgtagcatac catgatcgtg catgtaaata gtcatggtac 25080 tccagcctgg gcaacgtagt gagttactga tcaaaaaaaa agtgaagttt tctaaattaa 25140 agaactatgg taatggacat tattttctgt ttgcccttga tacaagaaaa gataaagaga 25200 gatagactgc cgggcgcggt ggctcatgcc tgtaatccca gcactttggg aggctgaggt 25260 gggcgaatca cctgaggtca ggagttcgag accagcctga ccaacatgga gaaaccctgt 25320 ctctactaaa aatacaaaat tagccaggcg tgatggccca tgcttgtaat cccagctact 25380 caggaagctg aggcaggaga atcgcttgaa cctgggaggc ggaggttgca gtgaaccgag 25440 attgtgccat tgcactccag cctgggcaac aagagcaaaa ctctgtctca aaaaaaagtg 25500 aaatggacta atacctcctt aggtcccttt gtagcttcac tcattgactc catcccttta 25560 ttgtttgttt gtttgtttgt ttatttattt atttaagatg gagtctcact ctgtcaccca 25620 ggctggagtg cagtagtgcg atctcagctc actgcaacct acacctcctg ggttcaagtg 25680 attctcctgc ctcagcctcc caagtagctg ggactacagg catgcaccac cacacccggc 25740 taattcttgt atttttagta gagatggggt ttcactatgt tggccaagct ggtcttgaac 25800 tccttacctg aagtgatcca cctgtcttgg cctccgaaag tgctaggatt acaggtgcga 25860 gccttggcgt caggctcctt tattgttatt ataatttggc atgggggacg gggtaggtct 25920 cactgtgttg cccaggctgg cgtacagtga tttgatcaca gctcactaca gcccccgaag 25980 tcctgggctc aagccatcct cccgccttgg cctcccaaag tgctaggatt acaggtgtga 26040 gccagtgcac cagcctgtcc ttttattatg atcctactta aatgcattct gaagaaaaaa 26100 taagtaaata ttgatactga acccttcaaa aatgatgttt ttggactgca ttgtttcttg 26160 tgttttattt actgacaaaa attattggtt gattagaaag gtaacttcca ggatttctaa 26220 tttttttttt ttttttcccc agacagagtc tcactctgtc gcccaggctg gagtgcagtg 26280 gtgcgatttc ggctcactgc aacctccgcc ccccaggttc aagtgattct cctgcctcag 26340 cctcctgagt agctgggatt acaggcgcct gtcaccacac ctggctaatt tttngtgttt 26400 ttagtagaga tgcggtttca ccgtcttctg caggctggtc ttgaacccct gaccttgtga 26460 tccacccgcc tcagcctccc aaagtgctgg gattacaggc atgagccatc acacctggcc 26520 tgatttctaa gttttatagg aactacaggc cgtgcctggt ggttcatgcc tgtaatttca 26580 gcactgtggg aggctgagat gggtggatca actttaggcc aggagtttga gaccagcctg 26640 gccaacctga tgaaaccccc atctctacaa aacatacaaa aaaattagcc aggcatggtg 26700 gtgcattcct gtaatgccat gtacgtggga ggctgaggca tgaaaatttc ttgaacctgg 26760 gaggtggaga ttgcagtgag tcgagatcac accactgcat tccagtctgg acagagtgag 26820 actctgcctc caataaaaaa ataaataaat aggctgggcg cagtgattca cacctgtaat 26880 cccacctgta atcctagtcc ttcgggaggc caagatgggc ggatcatgaa gtcaagagat 26940 cgagaccatc ctgaccaaca tggtgaaacc ccatctctac taaaaataca aaaaattagc 27000 cgggcatggt gatgggcgcc tgtagttcca gctacttggg tggctgaggc aggagaatgg 27060 tgtgaaccca ggaggtggag cttgcagtga gctgagattg cgccactgca ctccagcctg 27120 ggtgacagag caagactctg tctcaaaata aataaataaa tacataaata aataaataag 27180 ggaataacag catctgataa attatagctg acaaatattg caaaaattaa cctcttaaaa 27240 catactgtga ttcccatcat ttgtattact ttgaggaata ccctttttct ttcctatccc 27300 cagtttctcg atctttgggc atgaatgcat cagtgacaat tgaaggaaca tctgctgtgt 27360 acaaactccc aattgcacca ttaattatgg ggtcacatcc agttgacaat aaatggtaag 27420 ttaaatataa ttttagagta gcaaagtcaa tttttctcaa agcgcagtat gtaacaagtt 27480 ttaacctggc tgttacccca cttgaatttc tagaggttgg ccttttgttt tatccattca 27540 ttggtttctc ttttgtttga tatctgagtg ttcctctcca aagtcggctc gtagaacaca 27600 ttctctgcac tgaaagggaa agaagagaac tgtgctaatt cctctggctt aatgagtgcc 27660 cccttttatc ttacgttcat tagttacatt ggagtaataa ctggagaaca tgcctcagga 27720 taaatgtttg attaaattct acagtgttaa ccaatagcat gtctgggaat tgggggtagg 27780 aagtttcaca gagtgagctt ttgagagttt gtacttatgt gtctgtgtga tacatacatg 27840 tttaagaata ggtgtttgtg gccaggcatg gtggctcacg cctgtaatcc cagcactttg 27900 ggaggctgag gcaagtggaa cacctgaggt caggagttca agaccagcct ggccaacatg 27960 gtgaaacccc atctctacta acaatacaaa aattagccgg gcgtggtggc gggcacctgt 28020 agtcccagct actcacgaag ctgagatagg agaatccctt gaacccggga ggcagaggtt 28080 gcagtgagcc aagatcacgc cattgcactc tagcctgggc attaagaggg aaaatctgtc 28140 tcaaaaaaaa aaaaggtgtt tgggtttttc atttttatat caagtcagaa tccaaccctt 28200 ccaccctctt ttttttgaag gaccccttcc ttctcctcaa tcaccagtgc caacagtgtt 28260 gatcttcctg cctgtttctt cttgaaattt ccccagccaa tcccagtatc tagagcattt 28320 gttcagaaac tgcagaactg cacaggtgag ttttgagcat caggatctag tacttgttat 28380 aaatgtgaaa aagaaacttc aaagtcaggt tatcatgacg tctttcagtc ttacatgcct 28440 cattgaagta ttactggtaa agatgttata gtgctttgta ctagtaaatg cttagtaagt 28500 actcttttgg gtgaatgaat gtagatatca aggggaaact gagctgtcag tgttttcatc 28560 ttaacatgat cattatattg attcttgttt ttccaggaat tccattgttt gaaactcaac 28620 caacttatgc acccctgtat gaactgatca ctcagtttga gctatcaaag gaccctgacc 28680 ccataccttt gaatcacaac atgagatttt atgctgtaag taaagcccta gggagctaga 28740 atgttgtagg ttatgtttgg acccaagatc ataatcttcc tttatagcat tattgaggtc 28800 agataagttt cttcaagact gctggatcat gtcctatttc tttagtaagg caggaaatta 28860 cagaaatctc agaaatgggc tgggcgcggt ggctcacacc tgtaatcccc gcactttagg 28920 aggccaaggc aggcggatcg tgaggtcagg agttcgagac cagcctggcc aacataggga 28980 aaccctgtct ctactaaaaa tacaaaaaat tagccgtgtg tggtggcggg tgcctgtaat 29040 cccagctact cgggaggctg agggaggaga atggcttgaa tctgggaggc agaggttgca 29100 gtgagacaag attgtgccat tgtgctccag cctgggtgac agagcgagac tctgtctcaa 29160 aaaaaaaaaa aaaaaaaaga aatctcggaa atgtattgaa gtaaaagggc atgagcttcc 29220 attaggaaac aacaatgtac agttctttgt tgaaattttg ggtgattggc caggtgcagt 29280 ggctcacacc tgtaatccca ggaatttggg aggccaaggc gggtggtcac tagagatcag 29340 gagtttgaga ccagcctgga taacatggtg aaaccccgtc tctactaaaa atataaaaat 29400 tagctgggtg tggtggaaca tgcctgtaat cccagctact tgggaggctg aggcaggaga 29460 atcacttaac ccgggaggca gaggttgcag tgagccaaga ttgcgccact gcactccagc 29520 ctgggcaaca agagcgaaat tccgtctcaa aaaaaaaaaa aaaaaaaaaa aacgaggctg 29580 ggcgtggtgt ttcacatgtc taatcccagc agcactttgg gaggccgagg caggtggatc 29640 acgaggtcaa gagatcgaga gcatcctggc caacagggtg aaaccccatc tctactaaaa 29700 aaatacaaaa attagctggg tgtggtgtca tgcacctgta gtcccagcta ctcgggaggc 29760 tgaggcagga gaattgcttg aacccgggag acgaaggttg cagtgagcca aaatcgcacc 29820 actgcgcttc agcctggcaa cagagcaaga ctccgtctca aaaaaataaa aaagaaaaag 29880 aaattttggg tgatagactt tttttttttt ttttgagacg gagtcttgct gtgtcggcca 29940 ggctggagtc ctgtggtggg atcttcactc actgcaaact ccacctcccg ggttcatgcc 30000 attctcctgc ctcagcctcc tgagtagctg ggactacagg cgcctgccac catgcctggc 30060 taattttttg tatttttagt agagacggga tttcaccatg ttagccagga tggtctcgat 30120 ctcctgacct cgtgatccgc ccgcctcggc ctcccaaagt gctaggattg caggcgtgag 30180 ccaccttgcc tggcggggtg acagactctt aatggtattt tgaactttta gaatatcata 30240 attaggtgtt ataaattctt actgtttaat tctttcaagt gccatgagct tagaatctgg 30300 aaagatcctg aaagtactta cagagaggaa aatgctggaa gcagtaatta agaggctggg 30360 cacagtgact cacacctgta atcccagcac tttgggaggc caaggcaagg cagatcactt 30420 gagctcagga gttcaatacc agcctgggca acgtggcaaa atcctctctt ttttattttt 30480 ttattttttg agatggagtc tagctagctc tgttgccnag gctnggagtg cagtggcgcg 30540 atctcggctc actgcaacct ccgcctccca ggttcaagtg attctcctgc ctcagcctcc 30600 tgagtagctg ggactgtagg tgtgcaccac catgcccaga taatttttgt atttttagta 30660 gagacggggt ttntaccatg ttggccagat tggtctttat ctcttgacct tgagatccac 30720 cctactccgc ctcccaaagt gctgggatta caggcgcaag ccaccttgcc tgtccttttc 30780 ttttcttttt tctttttttt ttttttgctc ttggtgccca ggctggagtg cagtggcacg 30840 atctcggctg actgcaacct ctgcctccca ggttcaagcg attcttctgc ctcatcctcc 30900 ggcaaaatcc tgtctctaca aaaaaaaaat acaaaaatta ggctgatgtg gccgggagcg 30960 gtggctcacg cctgtaatcc tagcactttg ggaggccgag gcgggcagat catgaggtca 31020 ggagatcgag accatcctgg ccaacatggt gaaaccccgt cactactaaa aaacacaaaa 31080 aattagctgg gtgtggtggc gggcgcctgt agtcccagct acttgggagg ctgaggcagg 31140 agaatggcgt gaacccggga ggtggagctt gcagcgagca gagatcgtgc cactgcactc 31200 cagcctgagc tacaagagca agactctgtc tcaaaaaaaa aaaaaaaaaa aaataggcac 31260 atgtggttgt tcatgcctgt agtctcagct gctcgggaag ctgaggggct tcatcctggg 31320 aggtagagat tgcaatgagc caagattgca caattgtact ccatcctggg taatgggagt 31380 gaaaccctgt ctcaaaagta ataataataa taataaggga acgttccaaa acctcatctg 31440 gacttgtatt tatctgaatc ttttacaata gactaaagta gtggttacca aattctggct 31500 gcattattat tatataggtg ttttattaaa aatagagaat ttgcagccgg gcgtggttgt 31560 ttattcctgt aatcccagca ctttgggagg cccaggtggg tggatcacga agtcaggaga 31620 tggagacaat cctggctaac acagtgaaac actgtctcta ctaaaaatac aaaaaaaatt 31680 agccgggcgt ggtggcgggt gcctatagtc gcagctactc aggaggctga ggcaggagaa 31740 tggcatgaac tcgggaggcg gagcttgcag tgagccgaga tcgcaccact gcactccagc 31800 ctgggcaaca gagggagact ctgtctcaaa aaaaaaaaaa aaaaaaagag attttgcatt 31860 tatggtttag gaacctcttt aatacagatt tctgaggtag tttttatgtt agctaggtta 31920 ggaaatcact agcatagtgc atttactaaa aagacatctt ttcagcctaa ttagtaattg 31980 attgacattt ataatatcag ctgatgaaaa cattcactgt ggagactttt ctgcactttt 32040 ttgagtagta ggttttttca ggatactttg tggttggaat acaataggta actaaatagc 32100 actcgttaac tgttcttttt gcccaggctc ttcctggtca gcagcactgc tatttcctca 32160 acaaggatgc tcctcttcca gatggccgaa gtctacaggg aacccttgtt agcaaaatca 32220 cctttcagca ccctggccga gttcctctta tcctaaatct gatcagacac caagtggcct 32280 ataacaccct cattggaagc tgtgtcaaaa gaactattct gaaagaaggt actgcctttc 32340 ctttgatatg cttactggaa gatggaaact tacttctcca gtggttcttt aattggtggc 32400 ccatcagaat cacctgtaga acttaaaaaa agtttttttt ttttcatatt tggcataaaa 32460 atcaagcaaa ttagctgcac gtggtggtgc acgactgtag tcccacctac ttgggaggct 32520 gaggaaggag aatcgcttga acccgggagg cgggggttgc agtgagctta gatcatgcca 32580 ttgcactcca gcctgggtga cagagcaaga ctctgtctca aaaaaaaaaa aaaattcaaa 32640 catgtggagc ttttaacact acagatgatg gccaggtgtg gtggctcacg cctgtaatcc 32700 cagcactttg ggaggccaag gtgggcaaat cacttgagcc catgatttga gatcggtctg 32760 aggaacatgg caaaacccca tctctagaaa aaagtagcca gttgtggtgg tgtgcgcctg 32820 tggtcccagc tactcgggat cctgaggcag gaggattgtc gcttgagcct gggaggcaga 32880 gattgcagtg agctcagatt gcaccactgc actccaacct gggtaacaca gtaagaccct 32940 gaatcaaaaa caaaacacca ggctgagtgc agtggtttgt gcctgtaatc ccagcacttt 33000 gggaggccga ggtgggcaga tcacaaggtc aagagattga gaccatcctg gccaactagt 33060 gaaacccagt ctctactaaa aatacaaaaa ttagctgggt gtggtggtat acacctgtag 33120 tcccagctac ttgggagtct taggcaggag aatcacttga acccaggagg cggaggttgc 33180 actgagctta gatcatgcca ctgtactcca gtgtgggtga cagaatgaga ctccctctca 33240 aaaaaaaaaa aaaaatcaca gatgctgtgt ttccacacga agatgaactg aatgataatc 33300 tcagtgggaa gagttgggaa tctaaactcc acaaatcatt ttgatggaac cagtctgtag 33360 actagcattt ggagaccact gctttttgca atgttcttaa ctctttttct gaacatagta 33420 atgtaactgg gtattttagg ggctgtaccc tgtagcagat atacataagg tagaacttgg 33480 aatgctgtag tgagagaggc tttttttttt ttgagacagg gtctcactct gttgcccaga 33540 ctgaaccaca gtggcatgat aacagctttc tgctgcctct acctccttgg ctcaagcgat 33600 cctcctgcct tagcctccca agtaggtggg gctataggca tgtaccacca agcctggcta 33660 attttttctg tagagacagg gtctctctat gttgcccatg ttcgtctgta actcctgggc 33720 tgaagcgatc ctcttgacta ggcctcccaa ggtgctggga taataggtgc caccgtgctt 33780 ggccagtttt aggttaaatt ttaggttgtt aggagagtgc ttcccaggtt tagttctgag 33840 tatgttttct ctccccattg tttggagaac ttaatcttac agctttaaga aggtttagct 33900 cttttttctc tcaggattct ggaatgctga tgaatttttt tttttttttt tttttttttg 33960 agacggagtc tcactctgtt ccccaggctg gagtgcagtg gcatgatctt ggcccactgc 34020 aacctccgcc tcccaggttc aagcgattct cctgcgtcag cctctggcta attgtttaaa 34080 ttttttttag tagagatggg gtttcaccat catggccagg ctggtctcga actcctgacc 34140 tcgtgatcca ccctcctagg cctcccaaag tgctgggatt acaggcgtga gccatggcac 34200 ccggcgtaaa tgctgatgat tttcaaagac agttggctat tagaagcttt gctgtaaatt 34260 ttggctgttc tagctaacca tagttaattt taaaaatctt aagtcagaag cgctctgttg 34320 atagatatgt gtcagtgcca gtgatagaca ataagaaata gggaaggcaa atcagatctc 34380 ttttattttt atttatttat tttttgtttg ttttttgaga cggagtctcg ctctgtcgcc 34440 caggctggag tgcagtggcg caatttcggc tcactgcaag ctctgcctcc tgagttcacg 34500 tcattctcct gcctcagcct cccgagtagc tgggactaca ggcacccgcc atcacgcccg 34560 gctaattttt ttgtattttt agtagaaacg gggtgtcacc gtgttggcca ggatggtctc 34620 gatctcctga cctcgtgatc tgcccgcctg agcctcccag agtgctggga ttacaggcat 34680 gagccaccac gcctggcctt tttgtttttt tttttgagac ggagtcttgc ttggtcaccc 34740 aggctggagt gcagtggtgt gatcttcact cactgcaacc tttgcctcct gggttcaagc 34800 aattctcttg cttcagcctc ccaagtagct cggattacaa gtatccacca ccatgccagg 34860 ctaatctttg tagttttacc agagacgggg tttcgtcgag ttggccaggc tggttttgaa 34920 ctcgtgacct cagtggtctg cctgcctcgg cctcccaaag tgctgggatt acatgcgtga 34980 gccaccatgc ctggactttt ttttcttgat acagggtctt gctctgttat cgagattgga 35040 aagcagtggt acagtcatag ttcactttgg cctcaaactc ctgggctcaa gtgatactct 35100 tccctcaggc tcctcaagtc gctggactat aggcatgcac ctctacatct agtcaaattt 35160 taaagaaatt tatattatac tttttatttc agtgtacata tttatacagc aacatttttt 35220 ttgtagagat ggtgtcttgc catgttgtat aggatagtct tgagctcctg gcctcaggca 35280 ttcctcccac ctcagtgtcc caaagtgttg ggattacagg catgagccac tgtgctgggc 35340 caggagattc ttctcatgga aataaaaatt gctttagaaa tgagaaattt ttagtagttg 35400 gagtaataac atagagcata tatgttcaaa gttgtggcac ttctgtttca tgtgttaaaa 35460 tgccttaaga agtctccaga tagctttagg gcatttaaaa atgagttttc ataaggcagt 35520 tatggagcta atcctataga gaggcatgct ttttacaaag aaagtatatg atccttgtgt 35580 ctaggctgct agcacatctg gcataagaac atatgatgat ggctagatat atgtggaata 35640 aatacattat tttagattat agcctctata tctctgcttg ccaaactatg ggctgcacat 35700 ctaacagctg caaacacttg atagaaagca gagtttatgg cagtggttct ggatatctgc 35760 ttatcaagta gaatgagaca ctcggccact tttattttca gaattcactt ttacatttta 35820 gggattaagt ttcttcatct tggaaaattg aaagtctagc tacttttgtt ctggagaaat 35880 ttataacaaa ctggcaggtt ccttggaatc tgattccttc tgtcatctgt ggatgacata 35940 tagtaggttt tgtatgtgaa aaaagtgtgc gatttagttt ccaaggaggg agagagaatt 36000 tcagtctgta catttaagga atgttatttg tgtgaagtct tttttttttt tttttgagac 36060 agagtgttgc tctgtttccc aggctggggt tcagtgacat gatctcggct cactgcaacc 36120 cttgcctccc aggttgaaac gattctcctg tctcagccct ctgagtagct aggattacag 36180 gtgtgcgcca ccacacccgg ctaatttttt tttttttttt tttttttttt gagatggagt 36240 cttactcttg cccaggctgg agtgcagtgg cacgatctca gctcactaca acctccgcct 36300 cctaggttca agtgattctc ctgcctcagc ctcccgagta gctgggatta caggcatgtg 36360 ccaccacgcc cgactaattt tgtattttgt atttttagta gagatggggt ttgtccatgt 36420 tggtcaggct tgtctcgaac tcctgacctc aggtgatgag cccacctcag cctctcaaag 36480 ttctgggatt acaggcctga gccactgcat ccggcctctt ttttttattt tttagtggag 36540 ttggggtttc gccatgttgg ccaggctggt cttgaactcc tgacctcagg taatnccacc 36600 tgccttggcc tcccaaactg ctgggattac aggcgtgagc caccgtgccc agcctgtgtg 36660 aagtcttata tttggttgtt ttatgatctt tctctttgcc ttttagattc tcctgggctt 36720 ctccaatttg aagtgtgtcc tctctcagag tctcgtttca gcgtatcttt tcagcaccct 36780 gtgaatgact ccctggtgtg tggtgagttg tgtgggtggc taacacctct catggagcgg 36840 tttggtccct atgtcttttt tggaaaagtc agaagctaaa tctaaaacaa tttcctatgt 36900 attttgtctt tcagtggtaa tggatgtgca ggactcaaca catgtgagct gtaaactcta 36960 caaagggctg tcggatgcac tgatctgcac agatgacttc attgccaaag ttgttcaaag 37020 gtagccttgg ccctttttca tctgagtccc atttagagat gtataaagaa tgttgttgag 37080 tagggcgcgg tggctcacgc ctgtaatccc cacactttgg gaggccgagg caggcggatc 37140 acgaggtcag aagattgaga ccattctggc taacatggtg aacccccatc tctactaaaa 37200 atacaaaaat tagtcaggcg cgatggcggg cacatgtagt accagctact cgggaggctg 37260 atgcagaaga ataacttgaa cctgggaggt ggaggttgca gtgagccaag attgcgccat 37320 tgcactccag cctgacgaca gagcaagact ccgtgttttt gtttttgttg taaaaaaaaa 37380 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aggccgggcc tggtggctca cgcctgtaat 37440 cccagcactt tgggaggtga aggcgggtgg atcacgaggt caggagattg agaccatcct 37500 ggctaacaca gtgaaacccc gtctctatta aaaatacaaa aaattagccc ggcatggtgg 37560 cgggcgcctg tagtcccagc tactagggag gctgaggcag gagaatggca tgatcccggg 37620 aggtggagct tgcagtgagc cgagatcatg ccactgtact ccagcctggg caacagagtg 37680 agactctgtc tcaaaaaaaa aaaaaaatgt tgttttcaga attagcatga atttcaaata 37740 aaggagcaat ttagttattg ctttgatttc acccttctct gctttcggat tcaaagtaat 37800 ctaagctttg tttttttttt ttgagatgga gtcttgctct gatgcccaga ttgtacagtg 37860 gtacaatctt ggctcactgc aacctctgcc tcccgggttc aagtgattct cctgcctcac 37920 cctcctgagt agctaggatt acaggcatgt gctaccacac ctgactcatt tttgtatttt 37980 tagtagagac ggggtttcac catgttggcc aggctggtct cgaactcctg acctcaggtg 38040 atctgcctgc cttggcctcc caaagttctg ggattacagg cgtgagccac tgcacacagc 38100 cagctttgtt tttttttttt tttttttttt tttaatgata ttactatgag agtaatggga 38160 agtaaagtag gtaacttcag gcctggaatt atggtacttt aaggcaggag aaagccataa 38220 taaaatacag atatttttct tacctatcct gtgtgccaat tcttcataga tgtgacattt 38280 ttatcttttt attcttttat ttttttattt tgagacaaga gtctcactct gttacccagg 38340 ctggagtgca gtggtgtgat gacggctcac tgaagtgttg acctccgagg ttcaggttat 38400 ccttccacct agacctcctg agtagctggt actgcaggtg tgagccactg tgtctgcctg 38460 agcagatttc acaacttaaa tgttaggctg ggcgtggcag tgcatgcctg taatccagca 38520 ctttgggagg ccaaggcggg cagattactt gaggtcagga gttcgagacc agcctggcca 38580 acatgttgag atgtcatctc tataaaaata cgaaaattaa ccgagcatgg tggtatccgt 38640 ctgtcattct agctacttga gaggctgagg caggagaatc acttgaacct gggaatcgga 38700 ggttgcggtg agccaagatt gcgccactgt tctttagcct gggtgataga gtgagattct 38760 gtctcaaaac aaacaaacta actaaataaa taaaaatata aaaattagcc aggtgtggtg 38820 gcaggtgcct ataatcccag ctactcagga ggctgaggcg ggagaattgc ttgaacctgg 38880 gaggcggagg ctgcagtgag ccgagattgt gccactccac tccagcctgg tagacagagc 38940 aagatgccat ctcaaaacaa acaaaaagcc aacttaattg ttacgtgata gttaagccag 39000 tatgcatccc agaaccatta atatgtaatt ttttgagatt tttctacagc tgggtctggc 39060 agcctatttt tgtaaataaa ttttattgga acactgctgt tcatttacat gtctgtggca 39120 gatttctagt taatattgca gagtaggtga acaggaaaca agacctacaa agcctaacgg 39180 attactatat ggccttataa gaaaaatttg cggccgggtg cgggggtcac ccctgtaatc 39240 ctagcacttt gggaggctga ggcgggtaaa tcacgaggtc aggagttcaa gaccagcctg 39300 gccaagatgg tgaaacccca tctctactaa aaatacaaaa attagctggg cgtggtggcg 39360 ggcttctgta atcccagcta cttgggaggc tgaggcagag aattgcttga acctgggagg 39420 cggaagttgc ggtaagccaa gatcatgcca ctgcactcca gcctggataa cagagcgaga 39480 ctctgtctca aagaaaaaaa aaaaaaaaaa aagaaaaatt tgctggctgg gtgtggtggc 39540 tcacgcctat aatcccagca ctttgggagg tgggtggatc gcctgaggtc aggagttcga 39600 gaccagcctg actgacatgg agaaaccctg tctctactaa aaatacagaa ttagctgggc 39660 gtgatggcgc atggctgtaa tcccagctac tcgagaggct gaggcaggag aatcacttga 39720 acctgggagg tggaggttgc agtgagccaa gatcgcgcca ttgcactcca gcctgggcaa 39780 caagagcaaa actccgtctc caaaaaaaaa aaaaaaagaa aaaaaaattt gctgacatat 39840 gttctagagc aaaacgtttg caaactgagg tttatgaata taaatttatc tggaaaataa 39900 aaagtgcttt gctttaaaat aaacaagatt tataacagaa atagcacttt ttataatagt 39960 gtaggtttgg gatgatctaa gtcattaagg tttagtactt aatgggtcaa gagatttttc 40020 ttttcttttt tttttttttg agatggagtc ttgtactgtc acctgggctg gagtgccatg 40080 gcacaatctc agctcactgc aacctctgct tcccgggttc aagcaattct gcctcagcct 40140 cccaagtagt tgggattaca ggcacctgcc accatgccca gctaattttt tgtatttttt 40200 ctttttttta gtagagatgg ggtttcacta tgttggccag gctggtctca aaactcctga 40260 ccctgtgatc cacccgcctc ggcctcccaa agtgctggga ttacaggcgt gagccactct 40320 gaccggcgga gatttttcta atggttcctt aattaaaata aattatgtat ccctgtaatt 40380 accatatgaa gccccccaga ggacgacatt ttcttttttc ctttgaaatg aagtcttgca 40440 ctgtcaccaa ggctggcgtg caatgggacg atcttggctc gctgcgatct ctgcctcctg 40500 ggttcaagtg attctcctgt ctacccttcc tgagtagctg ggattacagc tgtgcaccgc 40560 cacggctggc taattttttg tatttttagt agagacaggg tttcaccccg ctggccaggc 40620 tggtctcaaa ctcctgaccc gaagtgatcc accctcctcg gccttccaaa gtgctgggat 40680 tgcaggagtg agccaccgcg cccgacctaa ggaggcattt ttttttttga gacggagtct 40740 cactctgttg ccaagctgga gtgcagtggt gtgatctcag tgcaatgcaa cctccgcctc 40800 ccttgttcaa gcaattctcc tgcctcaacc tcccgagtag ctgggattac aggcacgcgc 40860 caccacgccc agctaatttt tgtattttta gtagagacgg ggtttcacca tgttggccag 40920 gatggtgtcg atctcctgac ctcatgttcc agccgccttg gcctcccaaa gtgctgggat 40980 tacaggcgtg agccactgtg cccggcccct aaggaggcat tttcttactg agttccatta 41040 ttaccaattt tcttttagtt aagtttgaaa ttcaattatt tggactcttg tttatagttt 41100 gcttttgttg tgtgattcat aagaggttga caattttatg ttgggttaga tttttagaat 41160 gtatttaccc aaagctctgg tatttgatag aactattttt gtcttacaga ttatttgccc 41220 tacctgaaaa ctgcttactg caaaactctg atgttatatc taaatctcag gtctcattca 41280 gcagctattg caatgttaac tttgttttct tctttatttt gcagatgtat gtccatccct 41340 gtgacgatga gggctattcg gaggaaagct gaaaccattc aagccgacac cccagcactg 41400 tccctcattg cagagacagt tgaagacatg gtgaaaaaga acctgccccc ggctagcagc 41460 ccagggtatg gcatgaccac aggcaacaac ccaatgagtg gtaccactac accaaccaac 41520 acctttccgg ggggtcccat taccaccttg tttaatatga gcatgagcat caaagatcgg 41580 catgagtcgg tgggccatgg ggaggacttc agcaaggtgt ctcagaaccc aattcttacc 41640 agtttgttgc aaatcacagg gaacgggggg tctaccattg gctcgagtcc gacccctcct 41700 catcacacgc cgccacctgt ctcttcgatg gccggcaaca ccaagaacca cccgatgctc 41760 atgaaccttc ttaaagataa tcctgcccag gatttctcaa ccctttatgg aagcagccct 41820 ttagaaaggc agaactcctc ttccggctca ccccgcatgg aaatatgctc ggggagcaac 41880 aagaccaaga aaaagaagtc atcaagatta ccacctgaga aaccaaagca ccagactgaa 41940 gatgactttc agagggagct attttcaatg gatgttgact cacagaaccc tatctttgat 42000 gtcaacatga cagctgacac gctggatacg ccacacatca ctccagctcc aagccagtgt 42060 agcactcccc caacaactta cccacaacca gtacctcacc cccaacccag tattcaaagg 42120 atggtccgac tatccagttc agacagcatt ggcccagatg taactgacat cctttcagac 42180 attgcagaag aagcttctaa acttcccagc actagtgatg attgcccagc cattggcacc 42240 cctcttcgag attcttcaag ctctgggcat tctcagagta ccctgtttga ctctgatgtc 42300 tttcaaacta acaataatga aaatccatac actgatccag ctgatcttat tgcagatgct 42360 gctggaagcc ccagtagtga ctctcctacc aatcattttt ttcatgatgg agtagatttc 42420 aatcctgatt tattgaacag ccagagccaa agtggttttg gagaagaata ttttgatgaa 42480 agcagccaaa gtggggataa tgatgatttc aaaggatttg catctcaggc actaaatact 42540 ttgggggtgc caatgcttgg aggtgataat ggggagacca agtttaaggg caataaccaa 42600 gccgacacag ttgatttcag tattatttca gtagccggca aagctttagc tcctgcagat 42660 cttatggagc atcacagtgg tagtcagggt cctttactga ccactgggga cttagggaaa 42720 gaaaagactc aaaagagggt aaaggaaggc aatggcacca gtaatagtac tctctcgggg 42780 cccggattag acagcaaacc agggaagcgc agtcggaccc cttctaatga tgggaaaagc 42840 aaagataagc ctccaaagcg gaagaaggca gacactgagg gaaagtctcc atctcatagt 42900 tcttctaaca gaccttttac cccacctacc agtacaggtg gatctaaatc gccaggcagt 42960 gcaggaagat ctcagactcc cccaggtgtt gccacaccac ccattcccaa aatcactatt 43020 cagattccta agggaacagt gatggtgggc aagccttcct ctcacagtca gtataccagc 43080 agtggttctg tgtcttcctc aggcagcaaa agccaccata gccattcttc ctcctcttcc 43140 tcatctgctt ccacctcagg gaagatgaaa agcagtaaat cagaaggttc atcaagttcc 43200 aagttaagta gcagtatgta ttctagccag gggtcttctg gatctagcca gtccaaaaat 43260 tcatcccagt ctggggggaa gccaggctcc tctcccataa ccaagcatgg actgagcagt 43320 ggctctagca gcaccaagat gaaacctcaa ggaaagccat catcacttat gaatccttct 43380 ttaagtaaac caaacatatc cccttctcat tcaaggccac ctggaggctc tgacaagctt 43440 gcctctccaa tgaagcctgt tcctggaact cctnccatcc tctaaagcca agtcccctat 43500 cagttcaggt tctggtggtt ctcatatgtc tggaactagt tcaagctctg gcatgaagtc 43560 atcttcaggg ttaggatcct caggctcgtt gtcccagaaa actcccccat catctaattc 43620 ctgtacggca tcttcctcct ccttttcctc aagtggctct tccatgtcat cctctcagaa 43680 ccagcatggg agttctaaag gaaaatctcc cagcagaaac aagaagccgt ccttgacagc 43740 tgtcatagat aaactgaagc atggggttgt caccagtggc cctgggggtg aagacccact 43800 ggacggccag atgggggtga gcacaaattc ttccagccat cctatgtcct ccaaacataa 43860 catgtcagga ggagagtttc agggcaagcg tgagaaaagt gataaagaca aatcaaaggt 43920 ttccacctcc gggagttcag tggattcttc taagaagacc tcagagtcaa aaaatgtggg 43980 gagcacaggt gtggcaaaaa ttatcatcag taagcatgat ggaggctccc ctagcattaa 44040 agccaaagtg actttgcaga aacctgggga aagtagtgga gaagggctta ggcctcaaat 44100 ggcttcttct aaaaactatg gctctccact catcagtggt tccactccaa agcatgagcg 44160 tggctctccc agccatagta agtcaccagc atataccccc cagaatctgg acagtgaaag 44220 tgagtcaggc tcctccatag cagagaaatc ttatcagaat agtcccagct cagacgatgg 44280 tatccgacca cttccagaat acagcacaga gaaacataag aagcacaaaa aggaaaagaa 44340 gaaagtaaaa gacaaagata gggaccgaga ccgggacaaa gaccgagaca agaaaaaatc 44400 tcatagcatc aagccagaga gttggtccaa atcacccatc tcttcagacc agtccttgtc 44460 tatgacaagt aacacaatct tatctgcaga cagaccctca aggctcagcc cagactttat 44520 gattggggag gaagatgatg atcttatgga tgtggccctg attgggaatt aggaacctta 44580 tttcctaaaa gaaacagggc cagaggaaaa aaaactattg ataagtttat aggcaaacca 44640 ccataagggg tgagtcagac aggtctgatt tggttaagaa tcctaaatgg catggctttg 44700 acatcaagct gggtgaatta gaaaggcata tccagaccct attaaagaaa ccacagggtt 44760 tgattctggt taccaggaag tcttctttgt tcctgtgcca gaaagaaagt taaaatactt 44820 gcttaagaaa gggagggggg tgggaggggt gtagggagag ggaagggagg gaaacagttt 44880 tgtgggaaat attcatatat attttcttct ccctttttcc atttttaggc catgttttaa 44940 actcatttta gtgcatgtat atgaagggct gggcagaaaa tgaaaaagca atacattcct 45000 tgatgcattt gcatgaaggt tgttcaactt tgtttgaggt agttgtccgt ttgagtcatg 45060 ggcaaatgaa ggactttggt cattttggac acttaagtaa tgtttggtgt ctgtttctta 45120 ggagtgactg ggggagggaa gattatttta gctatttatt tgtaatattt taacccttta 45180 tctgtttgtt tttatacagt gtttcgttct aaatctatga ggtttagggt tcaaaatgat 45240 ggaaggccga agagcaaggc ttatatggtg gtagggagct tatagcttgt gctaatactg 45300 tagcatcaag cccaagcaaa ttagtcagag cccgccttta gagttaaata taatagaaaa 45360 accaaaatga tatttttatt ttaggagggt ttaaataggg ttcagagatc ataggaatat 45420 taggagttac ctctctgtgg aggtattgac ttgtaatctc attttccttt caaaaaaaaa 45480 aaaaaaagct aaggtggctt gttgggatgt aaacatgttt tcagatgcag taaggtttag 45540 tgtaggacag ccttcctgac ccagtggcat gaaaaccatt acaggattaa tagttctcct 45600 acttccacaa tgtgccaaaa gtctgcatcc cagcattttg tttgcaggag aactgatgcc 45660 attcctaaga gctggactca ctgtttctct tcatcacaag gagaaggagt ccaaacttta 45720 atcactccac tgtatgctcc ctgagataaa acagtaaaaa atccgcagcc atagttcact 45780 taaaacaatt tcaagctcac ttttgaagta atgggggcct ggaatgctag gtgagcatga 45840 agataaaccc ttgctactat gtagcaaccc aattggacct ttttggagaa ataggtctga 45900 gtctggattc tgggggacat caataagagc ccttcacata aaaatataga aatccaggag 45960 actgtttcga gtgcaacaga agttctcagt atttggaggg tcctctcaaa aattctgcgg 46020 ccttactttg atattgacac ctgcactgtg ccattcctga ttattccatt caggatctgt 46080 atcagcggga tggggcatgg tccccagcac aactcttctg ggttaaaaaa aaaaagccag 46140 gtgattcctt tgtgtgttat gtcgtaagtg gagtgacttc atcatatatg gaagaagatt 46200 tctatattca gctttttctg caggttggag tcagcataga gttggaaaat cagctttggc 46260 tttctttcct gtctcatttc ctctagtgtt ctccttttta ttgtcatcag ctctcaacaa 46320 ctctgccact tttgtgtccc aaggtaataa gatgtaggaa acaaaacatt gtaaagtgga 46380 gcaagaaaag ttatcaatta accacatcag agtcaaatgt cttgggtgac actaaggagg 46440 atatgggcag gtgataccag agtgctttat cttgtgatgt tgatacagta gcagcctctc 46500 agacattcag ccaggttgga tttctcatga gtttgtcacc tagttttgaa tcctatcccg 46560 ttggtttctg caggaaaaaa aaaaaaattt atgtggtttt taaaatttgt tctgagtggg 46620 gagaatctta gggggaatgt actgaatagt atcatgggct cagctccccc atgcagggcc 46680 aacaaacacc aaaatgagta aactgggaag cttttctctc tttctgtctt catcccagat 46740 caaagaatcc cgagttagga tctggatgaa ggataagccc ctgaattgtc gatgggctca 46800 cccccacact gacccagcat ctgaacttgc ttaacaggga gccggggcta aactgcttca 46860 ccctgcctga gaaccaggga gcactgcatt tctccacagg gtggaggaga agaggcagaa 46920 taaaccaagc ctgggacacc tnccctcctg tctaggtgta ctcattcttc tgtttcaaaa 46980 gaaggcaagg acatgaagtc aacttctacc tatcttctgc tgctggtgtc ttatgtattc 47040 tcagtttgac ctgattcctc ttctgtcttt tgacttaaca ttaggggttc ttggtcataa 47100 cctgctctga tgtacataaa gatttcaggt tcaatatcaa tgtgtcttaa aacagaagta 47160 ttttagcggg tggggggtgg ggtggtgggg acnaaacnaa cgcaggatat aattgccaaa 47220 accaggcttg aggttggtga ctcttgaaag attttctttc ttcaggccta gatcagaaaa 47280 ttaagtgcag caatatcatg aattctcaga agccctttca gggagccagt gagtcataca 47340 gtatccacag ttgagtcact taaagatgtc agtatacgaa acattattca caatccttgg 47400 gcaatctcat ttttttttcc ttctcccctc ctcccctgcc ccccatacat ttctatcctt 47460 gagttagttt tggaggggca ggaagtactt aacatctcag aagctagatt gggaaacatg 47520 ctcagctata agaactgagc tttaaatttt gagtttaaaa atgtacatca ggagcagctg 47580 gggagggtct ttttttaaaa aaatctttca aatttggttt tctgtgcata tggccgtttt 47640 gtaaatactt tggggttttt catttttttg aaagtagatg aaatctgttg tgggattttt 47700 ttcccgaaac attacaaaat aacctgttta tttacatgca aataaacttc tttgataaaa 47760 agtaatttgc ctgtgtgtaa taaaatttta gcctttgaat acttttttca tttgttcaaa 47820 actccagtca attttaaaga gtcagtcaac agtaaatgtc ttccctgagt actacttgaa 47880 atgggtcaac tggtaactga cggagctgtg aaacttgtct tcattctatt actctgtagg 47940 ccagtttttc taccctttgc catttttttt tttttcgaga tggagtctcg ctgtgtcgcc 48000 c 48001 <210> SEQ ID NO 5 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 5 aaagcagtaa atcagaaggt tcatca 26 <210> SEQ ID NO 6 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 6 ccagactggg atgaattttt gg 22 <210> SEQ ID NO 7 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 7 ttccaagtta agtagcagta tgtattctag c 31 <210> SEQ ID NO 8 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 8 gaaggtgaag gtcggagtc 19 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 9 gaagatggtg atgggatttc 20 <210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 10 caagcttccc gttctcagcc 20 <210> SEQ ID NO 11 <211> LENGTH: 5810 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (236)...(4936) <400> SEQUENCE: 11 gggaagatgg cggcggcctc gagcaccctc ctcttcttgc cgccggggac ttcagattga 60 tccttcccgg gaagagtagg gactgctggt gccctgcgtc ccgggatccc gagccaactt 120 gtttcctccg ttagtggtgg ggaagggctt atccttttgt ggcggatcta gcttctcctc 180 gccttcagga tgaaagctca ggggggaaac cgaggagtca gaaaagctga gtaag atg 238 Met 1 agt tct ctc ctg gaa cgg ctc cat gca aaa ttt aac caa aat aga ccc 286 Ser Ser Leu Leu Glu Arg Leu His Ala Lys Phe Asn Gln Asn Arg Pro 5 10 15 tgg agt gaa acc att aag ctt gtg cgt caa gtc atg gag aag agg gtt 334 Trp Ser Glu Thr Ile Lys Leu Val Arg Gln Val Met Glu Lys Arg Val 20 25 30 gtg atg agt tct gga ggg cat caa cat ttg gtc agc tgt ttg gag aca 382 Val Met Ser Ser Gly Gly His Gln His Leu Val Ser Cys Leu Glu Thr 35 40 45 ttg cag aag gct ctc aaa gta aca tct tta cca gca atg act gat cgt 430 Leu Gln Lys Ala Leu Lys Val Thr Ser Leu Pro Ala Met Thr Asp Arg 50 55 60 65 ttg gag tcc ata gca gga cag aat gga ctg ggc tct cat ctc agt gcc 478 Leu Glu Ser Ile Ala Gly Gln Asn Gly Leu Gly Ser His Leu Ser Ala 70 75 80 agt ggc act gaa tgt tac atc acg tca gat atg ttc tat gtg gaa gtg 526 Ser Gly Thr Glu Cys Tyr Ile Thr Ser Asp Met Phe Tyr Val Glu Val 85 90 95 cag tta gat cct gca gga cag ctt tgt gat gta aaa gtg gct cac cat 574 Gln Leu Asp Pro Ala Gly Gln Leu Cys Asp Val Lys Val Ala His His 100 105 110 ggg gag aat cct gtg agc tgt ccg gag ctt gta cag cag cta agg gaa 622 Gly Glu Asn Pro Val Ser Cys Pro Glu Leu Val Gln Gln Leu Arg Glu 115 120 125 aaa aat tct gat gaa ttt tct aag cac ctt aag ggc ctt gtt aat ctg 670 Lys Asn Ser Asp Glu Phe Ser Lys His Leu Lys Gly Leu Val Asn Leu 130 135 140 145 tat aac ctt cca ggg gac aac aaa ctg aag act aaa atg tac ttg gct 718 Tyr Asn Leu Pro Gly Asp Asn Lys Leu Lys Thr Lys Met Tyr Leu Ala 150 155 160 ctc caa tcc tta gaa caa gat ctt tct aaa atg gca att atg tac tgg 766 Leu Gln Ser Leu Glu Gln Asp Leu Ser Lys Met Ala Ile Met Tyr Trp 165 170 175 aaa gca act aat gct ggt ccc ttg gat aag att ctt cat gga agt gtt 814 Lys Ala Thr Asn Ala Gly Pro Leu Asp Lys Ile Leu His Gly Ser Val 180 185 190 ggc tat ctc aca cca agg agt ggg ggt cat tta atg aac ctg aag tac 862 Gly Tyr Leu Thr Pro Arg Ser Gly Gly His Leu Met Asn Leu Lys Tyr 195 200 205 tat gtc tct cct tct gac cta ctg gat gac aag act gca tct ccc atc 910 Tyr Val Ser Pro Ser Asp Leu Leu Asp Asp Lys Thr Ala Ser Pro Ile 210 215 220 225 att ttg cat gag aat aat gtt tct cga tct ttg ggc atg aat gca tca 958 Ile Leu His Glu Asn Asn Val Ser Arg Ser Leu Gly Met Asn Ala Ser 230 235 240 gtg aca att gaa gga aca tct gct gtg tac aaa ctc cca att gca cca 1006 Val Thr Ile Glu Gly Thr Ser Ala Val Tyr Lys Leu Pro Ile Ala Pro 245 250 255 tta att atg ggg tca cat cca gtt gac aat aaa tgg acc cct tcc ttc 1054 Leu Ile Met Gly Ser His Pro Val Asp Asn Lys Trp Thr Pro Ser Phe 260 265 270 tcc tca atc acc agt gcc aac agt gtt gat ctt cct gcc tgt ttc ttc 1102 Ser Ser Ile Thr Ser Ala Asn Ser Val Asp Leu Pro Ala Cys Phe Phe 275 280 285 ttg aaa ttt ccc cag cca atc cca gta tct aga gca ttt gtt cag aaa 1150 Leu Lys Phe Pro Gln Pro Ile Pro Val Ser Arg Ala Phe Val Gln Lys 290 295 300 305 ctg cag aac tgc aca gga att cca ttg ttt gaa act caa cca act tat 1198 Leu Gln Asn Cys Thr Gly Ile Pro Leu Phe Glu Thr Gln Pro Thr Tyr 310 315 320 gca ccc ctg tat gaa ctg atc act cag ttt gag cta tca aag gac cct 1246 Ala Pro Leu Tyr Glu Leu Ile Thr Gln Phe Glu Leu Ser Lys Asp Pro 325 330 335 gac ccc ata cct ttg aat cac aac atg aga ttt tat gct gct ctt cct 1294 Asp Pro Ile Pro Leu Asn His Asn Met Arg Phe Tyr Ala Ala Leu Pro 340 345 350 ggt cag cag cac tgc tat ttc ctc aac aag gat gct cct ctt cca gat 1342 Gly Gln Gln His Cys Tyr Phe Leu Asn Lys Asp Ala Pro Leu Pro Asp 355 360 365 ggc cga agt cta cag gga acc ctt gtt agc aaa atc acc ttt cag cac 1390 Gly Arg Ser Leu Gln Gly Thr Leu Val Ser Lys Ile Thr Phe Gln His 370 375 380 385 cct ggc cga gtt cct ctt atc cta aat ctg atc aga cac caa gtg gcc 1438 Pro Gly Arg Val Pro Leu Ile Leu Asn Leu Ile Arg His Gln Val Ala 390 395 400 tat aac acc ctc att gga agc tgt gtc aaa aga act att ctg aaa gaa 1486 Tyr Asn Thr Leu Ile Gly Ser Cys Val Lys Arg Thr Ile Leu Lys Glu 405 410 415 gat tct cct ggg ctt ctc caa ttt gaa gtg tgt cct ctc tca gag tct 1534 Asp Ser Pro Gly Leu Leu Gln Phe Glu Val Cys Pro Leu Ser Glu Ser 420 425 430 cgt ttc agc gta tct ttt cag cac cct gtg aat gac tcc ctg gtg tgt 1582 Arg Phe Ser Val Ser Phe Gln His Pro Val Asn Asp Ser Leu Val Cys 435 440 445 gtg gta atg gat gtg cag ggc tta aca cat gtg agc tgt aaa ctc tac 1630 Val Val Met Asp Val Gln Gly Leu Thr His Val Ser Cys Lys Leu Tyr 450 455 460 465 aaa ggg ctg tcg gat gca ctg atc tgc aca gat gac ttc att gcc aaa 1678 Lys Gly Leu Ser Asp Ala Leu Ile Cys Thr Asp Asp Phe Ile Ala Lys 470 475 480 gtt gtt caa aga tgt atg tcc atc cct gtg acg atg agg gct att cgg 1726 Val Val Gln Arg Cys Met Ser Ile Pro Val Thr Met Arg Ala Ile Arg 485 490 495 agg aaa gct gaa acc att caa gcc gac acc cca gca ctg tcc ctc att 1774 Arg Lys Ala Glu Thr Ile Gln Ala Asp Thr Pro Ala Leu Ser Leu Ile 500 505 510 gca gag aca gtt gaa gac atg gtg aaa aag aac ctg ccc ccg gct agc 1822 Ala Glu Thr Val Glu Asp Met Val Lys Lys Asn Leu Pro Pro Ala Ser 515 520 525 agc cca ggg tat ggc atg acc aca ggc aac aac cca atg agt ggt acc 1870 Ser Pro Gly Tyr Gly Met Thr Thr Gly Asn Asn Pro Met Ser Gly Thr 530 535 540 545 act aca tca acc aac acc ttt ccg ggg ggt ccc att gcc acc ttg ttt 1918 Thr Thr Ser Thr Asn Thr Phe Pro Gly Gly Pro Ile Ala Thr Leu Phe 550 555 560 aat atg agc atg agc atc aaa gat cgg cat gag tcg gtg ggc cat ggg 1966 Asn Met Ser Met Ser Ile Lys Asp Arg His Glu Ser Val Gly His Gly 565 570 575 gag gac ttc agc aag gtg tct cag aac cca att ctt acc agt ttg ttg 2014 Glu Asp Phe Ser Lys Val Ser Gln Asn Pro Ile Leu Thr Ser Leu Leu 580 585 590 caa atc aca ggg aac ggg ggg tct acc att ggc tcg agt ccg acc cct 2062 Gln Ile Thr Gly Asn Gly Gly Ser Thr Ile Gly Ser Ser Pro Thr Pro 595 600 605 cct cat cac acg ccg cca cct gtc tct tcg atg gcc ggc aac acc aag 2110 Pro His His Thr Pro Pro Pro Val Ser Ser Met Ala Gly Asn Thr Lys 610 615 620 625 aac cac ccg atg ctc atg aac ctt ctc aaa gat aat cct gcc cag gat 2158 Asn His Pro Met Leu Met Asn Leu Leu Lys Asp Asn Pro Ala Gln Asp 630 635 640 ttc tca acc ctt tat gga agc agc cct tta gaa agg cag aac tcc tct 2206 Phe Ser Thr Leu Tyr Gly Ser Ser Pro Leu Glu Arg Gln Asn Ser Ser 645 650 655 tcc ggc tca ccc cgc atg gaa ata tgc tcg ggg agc aac aag acc aag 2254 Ser Gly Ser Pro Arg Met Glu Ile Cys Ser Gly Ser Asn Lys Thr Lys 660 665 670 aaa aag aag tca tca aga tta cca cct gag aaa cca aag cac cag act 2302 Lys Lys Lys Ser Ser Arg Leu Pro Pro Glu Lys Pro Lys His Gln Thr 675 680 685 gaa gat gac ttt cag agg gag cta ttt tca atg gat gtt gac tca cag 2350 Glu Asp Asp Phe Gln Arg Glu Leu Phe Ser Met Asp Val Asp Ser Gln 690 695 700 705 aac cct atc ttt gat gtc aac atg aca gct gac acg ctg gat acg cca 2398 Asn Pro Ile Phe Asp Val Asn Met Thr Ala Asp Thr Leu Asp Thr Pro 710 715 720 cac atc act cca gct cca agc cag tgt agc act ccc cca aca act tac 2446 His Ile Thr Pro Ala Pro Ser Gln Cys Ser Thr Pro Pro Thr Thr Tyr 725 730 735 cca caa cca gta cct cac ccc caa ccc agt att caa agg atg gtc cga 2494 Pro Gln Pro Val Pro His Pro Gln Pro Ser Ile Gln Arg Met Val Arg 740 745 750 cta tcc agt tca gac agc att ggc cca gat gta act gac atc ctt tca 2542 Leu Ser Ser Ser Asp Ser Ile Gly Pro Asp Val Thr Asp Ile Leu Ser 755 760 765 gac att gca gaa gaa gct tct aaa ctt ccc agc act agt gat gat tgc 2590 Asp Ile Ala Glu Glu Ala Ser Lys Leu Pro Ser Thr Ser Asp Asp Cys 770 775 780 785 cca gcc att ggc acc cct ctt cga gat tct tca agc tct ggg cat tct 2638 Pro Ala Ile Gly Thr Pro Leu Arg Asp Ser Ser Ser Ser Gly His Ser 790 795 800 cag agt acc ctg ttt gac tct gat gtc ttt caa act aac aat aat gaa 2686 Gln Ser Thr Leu Phe Asp Ser Asp Val Phe Gln Thr Asn Asn Asn Glu 805 810 815 aat cca tac act gat cca gct gat ctt att gca gat gct gct gga agc 2734 Asn Pro Tyr Thr Asp Pro Ala Asp Leu Ile Ala Asp Ala Ala Gly Ser 820 825 830 ccc agt agt gac tct cct acc aat cat ttt ttt cat gat gga gta gat 2782 Pro Ser Ser Asp Ser Pro Thr Asn His Phe Phe His Asp Gly Val Asp 835 840 845 ttc aat cct gat tta ttg aac agc cag agc caa agt ggt ttt gga gaa 2830 Phe Asn Pro Asp Leu Leu Asn Ser Gln Ser Gln Ser Gly Phe Gly Glu 850 855 860 865 gaa tat ttt gat gaa agc agc caa agt ggg gat aat gat gat ttc aaa 2878 Glu Tyr Phe Asp Glu Ser Ser Gln Ser Gly Asp Asn Asp Asp Phe Lys 870 875 880 gga ttt gca tct cag gca cta aat act ttg ggg gtg cca atg ctt gga 2926 Gly Phe Ala Ser Gln Ala Leu Asn Thr Leu Gly Val Pro Met Leu Gly 885 890 895 ggt gat aat ggg gag acc aag ttt aag ggc aat aac caa gcc gac aca 2974 Gly Asp Asn Gly Glu Thr Lys Phe Lys Gly Asn Asn Gln Ala Asp Thr 900 905 910 gtt gat ttc agt att att tca gta gcc ggc aaa gct tta gct cct gca 3022 Val Asp Phe Ser Ile Ile Ser Val Ala Gly Lys Ala Leu Ala Pro Ala 915 920 925 gat ctt atg gag cat cac agt ggt agt cag ggt cct tta ctg acc act 3070 Asp Leu Met Glu His His Ser Gly Ser Gln Gly Pro Leu Leu Thr Thr 930 935 940 945 ggg gac tta ggg aaa gaa aag act caa aag agg gta aag gaa ggc aat 3118 Gly Asp Leu Gly Lys Glu Lys Thr Gln Lys Arg Val Lys Glu Gly Asn 950 955 960 ggc acc agt aat agt act ctc tcg ggg ccc gga tta gac agc aaa cca 3166 Gly Thr Ser Asn Ser Thr Leu Ser Gly Pro Gly Leu Asp Ser Lys Pro 965 970 975 ggg aag cgc agt cgg acc cct tct aat gat ggg aaa agc aaa gat aag 3214 Gly Lys Arg Ser Arg Thr Pro Ser Asn Asp Gly Lys Ser Lys Asp Lys 980 985 990 cct cca aag cgg aag aag gca gac act gag gga aag tct cca tct cat 3262 Pro Pro Lys Arg Lys Lys Ala Asp Thr Glu Gly Lys Ser Pro Ser His 995 1000 1005 agt tct tct aac aga cct ttt acc cca cct acc agt aca ggt gga tct 3310 Ser Ser Ser Asn Arg Pro Phe Thr Pro Pro Thr Ser Thr Gly Gly Ser 1010 1015 1020 1025 aaa tcg cca ggc agt gca gga aga tct cag act ccc cca ggt gtt gcc 3358 Lys Ser Pro Gly Ser Ala Gly Arg Ser Gln Thr Pro Pro Gly Val Ala 1030 1035 1040 aca cca ccc att ccc aaa atc act att cag att cct aag gga aca gtg 3406 Thr Pro Pro Ile Pro Lys Ile Thr Ile Gln Ile Pro Lys Gly Thr Val 1045 1050 1055 atg gtg ggc aag cct tcc tct cac agt cag tat acc agc agt ggt tct 3454 Met Val Gly Lys Pro Ser Ser His Ser Gln Tyr Thr Ser Ser Gly Ser 1060 1065 1070 gtg tct tcc tca ggc agc aaa agc cac cat agc cat tct tcc tcc tct 3502 Val Ser Ser Ser Gly Ser Lys Ser His His Ser His Ser Ser Ser Ser 1075 1080 1085 tcc tca tct gct tcc acc tca ggg aag atg aaa agc agt aaa tca gaa 3550 Ser Ser Ser Ala Ser Thr Ser Gly Lys Met Lys Ser Ser Lys Ser Glu 1090 1095 1100 1105 ggt tca tca agt tcc aag tta agt agc agt atg tat tct agc cag ggg 3598 Gly Ser Ser Ser Ser Lys Leu Ser Ser Ser Met Tyr Ser Ser Gln Gly 1110 1115 1120 tct tct gga tct agc cag tcc aaa aat tca tcc cag tct ggg ggg aag 3646 Ser Ser Gly Ser Ser Gln Ser Lys Asn Ser Ser Gln Ser Gly Gly Lys 1125 1130 1135 cca ggc tcc tct ccc ata acc aag cat gga ctg agc agt ggc tct agc 3694 Pro Gly Ser Ser Pro Ile Thr Lys His Gly Leu Ser Ser Gly Ser Ser 1140 1145 1150 agc acc aag atg aaa cct caa gga aag cca tca tca ctt atg aat cct 3742 Ser Thr Lys Met Lys Pro Gln Gly Lys Pro Ser Ser Leu Met Asn Pro 1155 1160 1165 tct tta agt aaa cca aac ata tcc cct tct cat tca agg cca cct gga 3790 Ser Leu Ser Lys Pro Asn Ile Ser Pro Ser His Ser Arg Pro Pro Gly 1170 1175 1180 1185 ggc tct gac aag ctt gcc tct cca atg aag cct gtt cct gga act cct 3838 Gly Ser Asp Lys Leu Ala Ser Pro Met Lys Pro Val Pro Gly Thr Pro 1190 1195 1200 cca tcc tct aaa gcc aag tcc cct atc agt tca ggt tct ggt ggt tct 3886 Pro Ser Ser Lys Ala Lys Ser Pro Ile Ser Ser Gly Ser Gly Gly Ser 1205 1210 1215 cat atg tct gga act agt tca agc tct ggc atg aag tca tct tca ggg 3934 His Met Ser Gly Thr Ser Ser Ser Ser Gly Met Lys Ser Ser Ser Gly 1220 1225 1230 tta gga tcc tca ggc tcg ttg tcc cag aaa act ccc cca tca tct aat 3982 Leu Gly Ser Ser Gly Ser Leu Ser Gln Lys Thr Pro Pro Ser Ser Asn 1235 1240 1245 tcc tgt acg gca tct tcc tcc tcc ttt tcc tca agt ggc tct tcc atg 4030 Ser Cys Thr Ala Ser Ser Ser Ser Phe Ser Ser Ser Gly Ser Ser Met 1250 1255 1260 1265 tca tcc tct cag aac cag cat ggg agt tct aaa gga aaa tct ccc agc 4078 Ser Ser Ser Gln Asn Gln His Gly Ser Ser Lys Gly Lys Ser Pro Ser 1270 1275 1280 aga aac aag aag ccg tcc ttg aca gct gtc ata gat aaa ctg aag cat 4126 Arg Asn Lys Lys Pro Ser Leu Thr Ala Val Ile Asp Lys Leu Lys His 1285 1290 1295 ggg gtt gtc acc agt ggc cct ggg ggt gaa gac cca ctg gac ggc cag 4174 Gly Val Val Thr Ser Gly Pro Gly Gly Glu Asp Pro Leu Asp Gly Gln 1300 1305 1310 atg ggg gtg agc aca aat tct tcc agc cat cct atg tcc tcc aaa cat 4222 Met Gly Val Ser Thr Asn Ser Ser Ser His Pro Met Ser Ser Lys His 1315 1320 1325 aac atg tca gga gga gag ttt cag ggc aag cgt gag aaa agt gat aaa 4270 Asn Met Ser Gly Gly Glu Phe Gln Gly Lys Arg Glu Lys Ser Asp Lys 1330 1335 1340 1345 gac aaa tca aag gtt tcc acc tcc ggg agt tca gtg gat tct tct aag 4318 Asp Lys Ser Lys Val Ser Thr Ser Gly Ser Ser Val Asp Ser Ser Lys 1350 1355 1360 aag acc tca gag tca aaa aat gtg ggg agc aca ggt gtg gca aaa att 4366 Lys Thr Ser Glu Ser Lys Asn Val Gly Ser Thr Gly Val Ala Lys Ile 1365 1370 1375 atc atc agt aag cat gat gga ggc tcc cct agc att aaa gcc aaa gtg 4414 Ile Ile Ser Lys His Asp Gly Gly Ser Pro Ser Ile Lys Ala Lys Val 1380 1385 1390 act ttg cag aaa cct ggg gaa agt agt gga gaa ggg ctt agg cct caa 4462 Thr Leu Gln Lys Pro Gly Glu Ser Ser Gly Glu Gly Leu Arg Pro Gln 1395 1400 1405 atg gct tct tct aaa aac tat ggc tct cca ctc atc agt ggt tcc act 4510 Met Ala Ser Ser Lys Asn Tyr Gly Ser Pro Leu Ile Ser Gly Ser Thr 1410 1415 1420 1425 cca aag cat gag cgt ggc tct ccc agc cat agt aag tca cca gca tat 4558 Pro Lys His Glu Arg Gly Ser Pro Ser His Ser Lys Ser Pro Ala Tyr 1430 1435 1440 acc ccc cag aat ctg gac agt gaa agt gag tca ggc tcc tcc ata gca 4606 Thr Pro Gln Asn Leu Asp Ser Glu Ser Glu Ser Gly Ser Ser Ile Ala 1445 1450 1455 gag aaa tct tat cag aat agt ccc agc tca gac gat ggt atc cga cca 4654 Glu Lys Ser Tyr Gln Asn Ser Pro Ser Ser Asp Asp Gly Ile Arg Pro 1460 1465 1470 ctt cca gaa tac agc aca gag aaa cat aag aag cac aaa aag gaa aag 4702 Leu Pro Glu Tyr Ser Thr Glu Lys His Lys Lys His Lys Lys Glu Lys 1475 1480 1485 aag aaa gta aaa gac aaa gat agg gac cga gac cgg gac aaa gac cga 4750 Lys Lys Val Lys Asp Lys Asp Arg Asp Arg Asp Arg Asp Lys Asp Arg 1490 1495 1500 1505 gac aag aaa aaa tct cat agc atc aag cca gag agt tgg tcc aaa tca 4798 Asp Lys Lys Lys Ser His Ser Ile Lys Pro Glu Ser Trp Ser Lys Ser 1510 1515 1520 ccc atc tct tca gac cag tcc ttg tct atg aca agt aac aca atc tta 4846 Pro Ile Ser Ser Asp Gln Ser Leu Ser Met Thr Ser Asn Thr Ile Leu 1525 1530 1535 tct gca gac aga ccc tca agg ctc agc cca gac ttt atg att ggg gag 4894 Ser Ala Asp Arg Pro Ser Arg Leu Ser Pro Asp Phe Met Ile Gly Glu 1540 1545 1550 gaa gat gat gat ctt atg gat gtg gcc ctg att ggg aat tag 4936 Glu Asp Asp Asp Leu Met Asp Val Ala Leu Ile Gly Asn * 1555 1560 1565 gaaccttatt tcctaaaaga aacagggcca gaggaaaaaa aactattgat aagtttatag 4996 gcaaaccacc ataaggggtg agtcagacag gtctgatttg gttaagaatc ctaaatggca 5056 tggctttgac atcaagctgg gtgaattaga aaggcatatc cagaccctat taaagaaacc 5116 acagggtttg attctggtta ccaggaagtc ttctttgttc ctgtgccaga aagaaagtta 5176 aaatacttgc ttaagaaagg gaggggggtg ggaggggtgt agggagaggg aagggaggga 5236 aacagttttg tgggaaatat tcatatatat tttcttctcc ctttttccat ttttaggcca 5296 tgttttaaac tcattttagt gcatgtatat gaagggctgg gcagaaaatg aaaaagcaat 5356 acattccttg atgcatttgc atgaaggttg ttcaactttg tttgaggtag ttgtccgttt 5416 gagtcatggg caaatgaagg actttggtca ttttggacac ttaagtaatg tttggtgtct 5476 gtttcttagg agtgactggg ggagggaaga ttattttagc tatttatttg taatatttta 5536 accctttatc tgtttgtttt tatacagtgt ttcgttctaa atctatgagg tttagggttc 5596 aaaatgatgg aaggccgaag agcaaggctt atatggtggt agggagctta tagcttgtgc 5656 taatactgta gcatcaagcc caagcaaatt agtcagagcc cgcctttaga gttaaatata 5716 atagaaaaac caaaatgata tttttatttt aggagggttt aaatagggtt cagagatcat 5776 aggaatatta ggagttacct ctctgtggag gtat 5810 <210> SEQ ID NO 12 <220> FEATURE: <400> SEQUENCE: 12 000 <210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 13 ctagatccgc cacaaaagga 20 <210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 14 gagagaactc atcttactca 20 <210> SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 15 ttaaattttg catggagccg 20 <210> SEQ ID NO 16 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 16 tgacgcacaa gcttaatggt 20 <210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 17 tccaaacagc tgaccaaatg 20 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 18 atgtctccaa acagctgacc 20 <210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 19 gttactttga gagccttctg 20 <210> SEQ ID NO 20 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 20 aagatgttac tttgagagcc 20 <210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 21 aaacgatcag tcattgctgg 20 <210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 22 agagcccagt ccattctgtc 20 <210> SEQ ID NO 23 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 23 tcagtgccac tggcactgag 20 <210> SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 24 atatctgacg tgatgtaaca 20 <210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 25 agaacatatc tgacgtgatg 20 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 26 cacatagaac atatctgacg 20 <210> SEQ ID NO 27 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 27 acttccacat agaacatatc 20 <210> SEQ ID NO 28 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 28 actgcacttc cacatagaac 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 29 tcacaaagct gtcctgcagg 20 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 30 tccccatggt gagccacttt 20 <210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 31 gattctcccc atggtgagcc 20 <210> SEQ ID NO 32 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 32 cacaggattc tccccatggt 20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 33 cagctcacag gattctcccc 20 <210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 34 gaagaatctt atccaaggga 20 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 35 gttccttcaa ttgtcactga 20 <210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 36 aatggaattc ctgtgcagtt 20 <210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 37 tagttctttt gacacagctt 20 <210> SEQ ID NO 38 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 38 caccatgtct tcaactgtct 20 <210> SEQ ID NO 39 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 39 atctttgatg ctcatgctca 20 <210> SEQ ID NO 40 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 40 tggtaagaat tgggttctga 20 <210> SEQ ID NO 41 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 41 gttccctgtg atttgcaaca 20 <210> SEQ ID NO 42 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 42 cccccgttcc ctgtgatttg 20 <210> SEQ ID NO 43 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 43 cgagccaatg gtagaccccc 20 <210> SEQ ID NO 44 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 44 ggactcgagc caatggtaga 20 <210> SEQ ID NO 45 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 45 gggtcggact cgagccaatg 20 <210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 46 tcgaagagac aggtggcggc 20 <210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 47 ggccatcgaa gagacaggtg 20 <210> SEQ ID NO 48 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 48 ttgccggcca tcgaagagac 20 <210> SEQ ID NO 49 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 49 tggtgttgcc ggccatcgaa 20 <210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 50 gttcttggtg ttgccggcca 20 <210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 51 atcgggtggt tcttggtgtt 20 <210> SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 52 tgagcatcgg gtggttcttg 20 <210> SEQ ID NO 53 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 53 gttcatgagc atcgggtggt 20 <210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 54 agaaggttca tgagcatcgg 20 <210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 55 ccataaaggg ttgagaaatc 20 <210> SEQ ID NO 56 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 56 tgcttccata aagggttgag 20 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 57 aagggctgct tccataaagg 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 58 ttctaaaggg ctgcttccat 20 <210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 59 tgcctttcta aagggctgct 20 <210> SEQ ID NO 60 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 60 agttctgcct ttctaaaggg 20 <210> SEQ ID NO 61 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 61 agaggagttc tgcctttcta 20 <210> SEQ ID NO 62 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 62 ccggaagagg agttctgcct 20 <210> SEQ ID NO 63 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 63 ctacactggc ttggagctgg 20 <210> SEQ ID NO 64 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 64 ccaatgctgt ctgaactgga 20 <210> SEQ ID NO 65 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 65 ctgggccaat gctgtctgaa 20 <210> SEQ ID NO 66 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 66 tacatctggg ccaatgctgt 20 <210> SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 67 tcagttacat ctgggccaat 20 <210> SEQ ID NO 68 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 68 atcaggattg aaatctactc 20 <210> SEQ ID NO 69 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 69 atgggagagg agcctggctt 20 <210> SEQ ID NO 70 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 70 tgagaggatg acatggaaga 20 <210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 71 ttatctatga cagctgtcaa 20 <210> SEQ ID NO 72 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 72 tgactcactt tcactgtcca 20 <210> SEQ ID NO 73 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 73 gagcctgact cactttcact 20 <210> SEQ ID NO 74 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 74 tggaggagcc tgactcactt 20 <210> SEQ ID NO 75 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 75 tgctatggag gagcctgact 20 <210> SEQ ID NO 76 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 76 taaggttcct aattcccaat 20 <210> SEQ ID NO 77 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 77 tatatgaata tttcccacaa 20 <210> SEQ ID NO 78 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 78 atacatgcac taaaatgagt 20 <210> SEQ ID NO 79 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 79 ctcctaagaa acagacacca 20 <210> SEQ ID NO 80 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 80 agtcacttac cctcggtttc 20 <210> SEQ ID NO 81 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 81 agagacaggg ttttaccttg 20 <210> SEQ ID NO 82 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 82 tgaagaagtt tctctctgaa 20 <210> SEQ ID NO 83 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 83 cttcagtttg ctggagagta 20 <210> SEQ ID NO 84 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 84 ataaagggat ggagtcaatg 20 <210> SEQ ID NO 85 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 85 aatggaattc ctggaaaaac 20 <210> SEQ ID NO 86 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 86 tacactaaac cttactgcat 20 <210> SEQ ID NO 87 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 87 gagcttgaaa ttgttttaag 20 <210> SEQ ID NO 88 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 88 cccactcaga acaaatttta 20 <210> SEQ ID NO 89 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 89 agataggtag aagttgactt 20 <210> SEQ ID NO 90 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 90 actttaggcc aaaagtgccc 20 <210> SEQ ID NO 91 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 91 tccttttgtg gcggatctag 20 <210> SEQ ID NO 92 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 92 cggctccatg caaaatttaa 20 <210> SEQ ID NO 93 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 93 accattaagc ttgtgcgtca 20 <210> SEQ ID NO 94 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 94 catttggtca gctgtttgga 20 <210> SEQ ID NO 95 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 95 ggtcagctgt ttggagacat 20 <210> SEQ ID NO 96 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 96 cagaaggctc tcaaagtaac 20 <210> SEQ ID NO 97 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 97 gacagaatgg actgggctct 20 <210> SEQ ID NO 98 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 98 tgttacatca cgtcagatat 20 <210> SEQ ID NO 99 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 99 catcacgtca gatatgttct 20 <210> SEQ ID NO 100 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 100 cgtcagatat gttctatgtg 20 <210> SEQ ID NO 101 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 101 gttctatgtg gaagtgcagt 20 <210> SEQ ID NO 102 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 102 cctgcaggac agctttgtga 20 <210> SEQ ID NO 103 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 103 aaagtggctc accatgggga 20 <210> SEQ ID NO 104 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 104 ggctcaccat ggggagaatc 20 <210> SEQ ID NO 105 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 105 accatgggga gaatcctgtg 20 <210> SEQ ID NO 106 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 106 ggggagaatc ctgtgagctg 20 <210> SEQ ID NO 107 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 107 tcagtgacaa ttgaaggaac 20 <210> SEQ ID NO 108 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 108 aagctgtgtc aaaagaacta 20 <210> SEQ ID NO 109 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 109 agacagttga agacatggtg 20 <210> SEQ ID NO 110 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 110 tgagcatgag catcaaagat 20 <210> SEQ ID NO 111 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 111 tcagaaccca attcttacca 20 <210> SEQ ID NO 112 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 112 tgttgcaaat cacagggaac 20 <210> SEQ ID NO 113 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 113 caaatcacag ggaacggggg 20 <210> SEQ ID NO 114 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 114 gggggtctac cattggctcg 20 <210> SEQ ID NO 115 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 115 tctaccattg gctcgagtcc 20 <210> SEQ ID NO 116 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 116 cattggctcg agtccgaccc 20 <210> SEQ ID NO 117 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 117 ttcgatggcc ggcaacacca 20 <210> SEQ ID NO 118 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 118 tggccggcaa caccaagaac 20 <210> SEQ ID NO 119 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 119 aacaccaaga accacccgat 20 <210> SEQ ID NO 120 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 120 accacccgat gctcatgaac 20 <210> SEQ ID NO 121 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 121 ccgatgctca tgaaccttct 20 <210> SEQ ID NO 122 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 122 ctcaaccctt tatggaagca 20 <210> SEQ ID NO 123 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 123 cctttatgga agcagccctt 20 <210> SEQ ID NO 124 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 124 atggaagcag ccctttagaa 20 <210> SEQ ID NO 125 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 125 agcagccctt tagaaaggca 20 <210> SEQ ID NO 126 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 126 ccctttagaa aggcagaact 20 <210> SEQ ID NO 127 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 127 tagaaaggca gaactcctct 20 <210> SEQ ID NO 128 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 128 aggcagaact cctcttccgg 20 <210> SEQ ID NO 129 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 129 ccagctccaa gccagtgtag 20 <210> SEQ ID NO 130 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 130 tccagttcag acagcattgg 20 <210> SEQ ID NO 131 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 131 ttcagacagc attggcccag 20 <210> SEQ ID NO 132 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 132 acagcattgg cccagatgta 20 <210> SEQ ID NO 133 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 133 attggcccag atgtaactga 20 <210> SEQ ID NO 134 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 134 gagtagattt caatcctgat 20 <210> SEQ ID NO 135 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 135 tcttccatgt catcctctca 20 <210> SEQ ID NO 136 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 136 tggacagtga aagtgagtca 20 <210> SEQ ID NO 137 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 137 agtgaaagtg agtcaggctc 20 <210> SEQ ID NO 138 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 138 aagtgagtca ggctcctcca 20 <210> SEQ ID NO 139 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 139 agtcaggctc ctccatagca 20 <210> SEQ ID NO 140 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 140 attgggaatt aggaacctta 20 <210> SEQ ID NO 141 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 141 ttgtgggaaa tattcatata 20 <210> SEQ ID NO 142 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 142 actcatttta gtgcatgtat 20 <210> SEQ ID NO 143 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 143 tggtgtctgt ttcttaggag 20 <210> SEQ ID NO 144 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 144 ttcagagaga aacttcttca 20 <210> SEQ ID NO 145 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 145 cattgactcc atccctttat 20 <210> SEQ ID NO 146 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 146 atgcagtaag gtttagtgta 20 <210> SEQ ID NO 147 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 147 taaaatttgt tctgagtggg 20 <210> SEQ ID NO 148 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 148 aagtcaactt ctacctatct 20 

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targeted to a nucleic acid molecule encoding PPAR binding protein, wherein said compound specifically hybridizes with said nucleic acid molecule encoding PPAR binding protein (SEQ ID NO: 4) and inhibits the expression of PPAR binding protein.
 2. The compound of claim 1 comprising 12 to 50 nucleobases in length.
 3. The compound of claim 2 comprising 15 to 30 nucleobases in length.
 4. The compound of claim 1 comprising an oligonucleotide.
 5. The compound of claim 4 comprising an antisense oligonucleotide.
 6. The compound of claim 4 comprising a DNA oligonucleotide.
 7. The compound of claim 4 comprising an RNA oligonucleotide.
 8. The compound of claim 4 comprising a chimeric oligonucleotide.
 9. The compound of claim 4 wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.
 10. The compound of claim 1 having at least 70% complementarity with a nucleic acid molecule encoding PPAR binding protein (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of PPAR binding protein.
 11. The compound of claim 1 having at least 80% complementarity with a nucleic acid molecule encoding PPAR binding protein (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of PPAR binding protein.
 12. The compound of claim 1 having at least 90% complementarity with a nucleic acid molecule encoding PPAR binding protein (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of PPAR binding protein.
 13. The compound of claim 1 having at least 95% complementarity with a nucleic acid molecule encoding PPAR binding protein (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of PPAR binding protein.
 14. The compound of claim 1 having at least one modified internucleoside linkage, sugar moiety, or nucleobase.
 15. The compound of claim 1 having at least one 2′-O-methoxyethyl sugar moiety.
 16. The compound of claim 1 having at least one phosphorothioate internucleoside linkage.
 17. The compound of claim 1 having at least one 5-methylcytosine.
 18. A method of inhibiting the expression of PPAR binding protein in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of PPAR binding protein is inhibited.
 19. A method of screening for a modulator of PPAR binding protein, the method comprising the steps of: a. contacting a preferred target segment of a nucleic acid molecule encoding PPAR binding protein with one or more candidate modulators of PPAR binding protein, and b. identifying one or more modulators of PPAR binding protein expression which modulate the expression of PPAR binding protein.
 20. The method of claim 19 wherein the modulator of PPAR binding protein expression comprises an oligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide, an RNA oligonucleotide, an RNA oligonucleotide having at least a portion of said RNA oligonucleotide capable of hybridizing with RNA to form an oligonucleotide-RNA duplex, or a chimeric oligonucleotide.
 21. A diagnostic method for identifying a disease state comprising identifying the presence of PPAR binding protein in a sample using at least one of the primers comprising SEQ ID NOs: 5 or 6, or the probe comprising SEQ ID NO:
 7. 22. A kit or assay device comprising the compound of claim
 1. 23. A method of treating an animal having a disease or condition associated with PPAR binding protein comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of PPAR binding protein is inhibited.
 24. The method of claim 23 wherein the disease or condition is a metabolic disorder. 